Translational Elements Research Articles

Structure and function of the Salmonella typhimurium and Escherichia coli K-12 histidine operons.

We have determined the complete nucleotide sequence of the histidine operons of Escherichia coli and of Salmonella typhimurium. This structural information enabled us to investigate the expression and organization of the histidine operon. The proteins coded by each of the putative histidine cistrons were identified by subcloning appropriate DNA fragments and by analyzing the polypeptides synthesized in minicells. A structural comparison of the gene products was performed. The histidine messenger RNA molecules produced in vivo and the internal transcription initiation sites were identified by Northern blot analysis and S1 nuclease mapping. A comparative analysis of the different transcriptional and translational control elements within the two operons reveals a remarkable preservation for most of them except for the intercistronic region between the first (hisG) and second (hisD) structural genes and for the rho-independent terminator of transcription at the end of the operon. Overall, the operon structure is very compact and its expression appears to be regulated at several levels.

Cis-acting translational effects of the 5' noncoding region of c-myc mRNA.

We have previously shown that the 5' noncoding region of mouse c-myc mRNA has a negative effect on translational efficiency in a rabbit reticulocyte lysate (A. Darveau, J. Pelletier, and N. Sonenberg, Proc. Natl. Acad. Sci. USA 82:2315-2319, 1985). We wanted to localize and characterize the inhibitory translational element(s) in the mRNA and to study its effect in other in vitro and in vivo systems. Here we report that the restrictive element is confined to a 240-nucleotide sequence of the 5' noncoding region of mouse c-myc mRNA and that this sequence acts in cis to inhibit the translation of a heterologous mRNA. In addition, we report that the cis-inhibitory effect is also exhibited in microinjected Xenopus oocytes and wheat-germ extracts but not in HeLa cell extracts. Transfection of corresponding plasmid DNA constructs into several established cell lines did not produce the cis-inhibitory effect. A model to explain these results is presented.

Cis-acting translational effects of the 5' noncoding region of c-myc mRNA.

We have previously shown that the 5' noncoding region of mouse c-myc mRNA has a negative effect on translational efficiency in a rabbit reticulocyte lysate (A. Darveau, J. Pelletier, and N. Sonenberg, Proc. Natl. Acad. Sci. USA 82:2315-2319, 1985). We wanted to localize and characterize the inhibitory translational element(s) in the mRNA and to study its effect in other in vitro and in vivo systems. Here we report that the restrictive element is confined to a 240-nucleotide sequence of the 5' noncoding region of mouse c-myc mRNA and that this sequence acts in cis to inhibit the translation of a heterologous mRNA. In addition, we report that the cis-inhibitory effect is also exhibited in microinjected Xenopus oocytes and wheat-germ extracts but not in HeLa cell extracts. Transfection of corresponding plasmid DNA constructs into several established cell lines did not produce the cis-inhibitory effect. A model to explain these results is presented.

Nucleotide and deduced amino acid sequence of hemagglutinin-neuraminidase genes of human type 3 parainfluenza viruses isolated from 1957 to 1983

We have sequenced the coding and noncoding regions of the hemagglutinin-neuraminidase (HN) genes of six clinical strains of human type 3 parainfluenza virus (PIV3) isolated between 1973 and 1983, and compared them to the prototype 1957 strain. Sequence variability does not result from the accumulation of mutations over time, but represents genetic heterogeneity in HN genes within the PIV3 population. Most of the nucleotide diversity occurs in the 5′ noncoding sequences, exclusive of regions supplying transcriptional and translational control elements. Although the overall amino acid homology among HN proteins is very high, most variability is concentrated in domains at the carboxyl and amino terminus. This uneven distribution of amino acid diversity may reflect both functional and structural constraints on different HN domains and the epidemiologic features of PIV3 infection.

Expression of domains of mouse alpha-fetoprotein in Escherichia coli

A mouse alpha-fetoprotein complementary DNA containing the coding region for amino acids 256 to 548 was fused to the lac transcriptional and translational control elements contained on the expression vector pOP203-28. The expression of a 35 kD hybrid lac Z′- alpha-fetoprotein polypeptide in Escherichia coli was demonstrated by the chloramphenicol release assay and by immunoprecipitation using rabbit anti-mouse alpha-fetoprotein antibodies as probe.

Response of a NPP reactor building under seismic action with regard to different soil properties

The object of this investigation is the response of a reactor building on seismic action with systematic variation of the soil stiffness. A thin-walled orthotropic containment shell on varying heavy and rigid foundations is regarded as calculation model. The soil stiffness is simulated by means of spring elements for horizontal translation and for rocking motions of the building. By the response spectra method the loads of the containment shell are calculated for a horizontal seismic excitation. The investigation is aimed at determining the influence of differentiated soil stiffnesses on the containment action effects and at recognizing the causes for the occurring effects. The results are thoroughly represented by selected quantities of the building's response, the effects from the soil-structure interaction are discussed and the causes of the effects clearly explained. A possibility is provided for determining critical soil stiffnesses which cause a significant intensification effect. The results of the investigations show that both the soil stiffness and structural configuration of the reactor building, particularly in case of the substructure being heavy and rigid, exert a decisive influence on the loading of the superstructure.

A comparison of bovine growth hormone expression directed by bGH genomic or intronless DNA in transiently transfected eukaryotic cells

Two recombinant DNA plasmids were constructed with identical transcriptional and translational regulatory elements controlling expression of the bovine growth hormone (bGH) gene or the bGH gene lacking introns. Transient expression of these plasmids in cultured eukaryotic cells, monitored by assaying secretion of bGH into the culture medium, was employed to examine the relative importance of introns in the expression of this gene. The bGH gene lacking introns is expressed more efficiently than the bGH gene in avian and mammalian cells.

Translational and transcriptional control elements in the untranslated leader of the heat-shock gene hsp22

Downstream of the transcription start site in the Drosophila heat-shock gene hsp22, we have identified a region that is necessary for efficient transcription, and also for selective translation during heat shock. We assayed the expression of mutated genes after P-factor-mediated insertion into the genome. Deletions within the first 26 nucleotides block the preferential translation of hsp22 mRNA at high temperature. The rate of transcription is also decreased, though transcription is still heat-inducible. Leaving this region intact, up to 86% of the leader can be deleted without affecting translation or transcription. The functional region coincides with a region of sequence homology between the heat-shock genes. Only partial homology is found to a conserved sequence, ATCA G TT C T, found at the very 5′ end of other insect genes.

Anisotropic thermal-parameter refinement of the DNA dodecamer CGCGAATTCGCG by the segmented rigid-body method

A structure-factor least-squares refinement of the deoxyoligonucleotide (CGCGAATTCGCG)2 has been conducted using a model with constraints and restraints on the positional parameters and a segmented rigid-body representation for the anisotropic temperature factors. The macromolecule was divided into subgroups each of which was treated as a rigid body in terms of both positional and thermal parameters. For each subgroup, the thermal parameters determined were elements of translation, libration and correlation (TLS) tensors. This segmented rigidbody model of thermal motion has not previously been applied to the refinement of a macromolecular crystal structure. The anisotropic thermal-parameter refinement has significantly reduced the classical R factor as judged by the Hamilton test. The resulting difference Fourier map has a considerably lower noise level allowing fifteen additional low-occupancy water positions to be identified. In addition, analysis of the anisotropic thermal parameters has revealed new information about the local mobility of the groups in the oligonucleotide. Thus, the method of segmented rigid-body anisotropic temperature-factor refinement appears to be uniquely suited to macromolecules, especially nucleic acids, where high-resolution data are usually unavailable.

Rapid mutational analysis of regulatory loci in Escherichia coli K-12 using bacteriophage M13.

A derivative of bacteriophage M13mp8 , designated M13mp8 /P, was prepared in which the promoter and NH2-terminal codons of bacterial genes may be fused to a portion of beta-galactosidase, resulting in an easily scorable phenotype. Because transcription from the inserted promoter remains responsive to the host regulatory system, it is simple to screen mutagenized phage for isolates with aberrant regulatory phenotypes and to determine the mutational changes by dideoxy sequence analysis. The feasibility of the method was demonstrated by isolation of a large number of mutations in the regulatory regions of two genes, lexA and recA. Base substitutions that altered the phenotype of recombinant phage were identified both in the single LexA repressor binding site of recA and in the two binding sites of lexA, as well as in other sites that likely affect translational efficiency. Our results suggest that this approach will be generally useful for mutational analysis of transcriptional and translational regulatory elements.

マルチストロボ装置を応用した下顎運動の研究 ア音発音時の下顎頭について

Many reports have been made by researchers in the past on the study of the mandibular movement, which is a matter of primary concern in prosthodontics. It has been made clear by these reports that the movements of mandibular condyle can be divided into two kinetic elements of translation and rotation. However, almost no report has been made so far concerning in what temporal process these two elements occur at the time of physiologic condyle movement, especially when phonation is made.Therefore, the author particulary took up the phonation of the [a:] sound, which is least influenced by occlusion and the vertical and horizontal overlap of the anterior. The author then paid attention to the movement of mandibular condyle when the [a:] sound was pronounced. And in order to serve for the diagnosis of dysfunction of the stomatognathic system, the subjects used for the present experiment were limited to those who had no abnormality in their stomatognathic system. By photographic analysis using a multiple stroboscope apparatus, and considering the time element, the author recorded and analyzed the translation and rotation of mandibular condyle, and reached the following conclusion.1) All the seven subjects tended to show very little difference in the amount of translation and the degree of rotation of their mandibular condyle, as well as in the time required until it came to the position ready to pronounce the [a:] sound.2) The amount of translation of mandibular condyle from the position of maximal intercuspal occlusion to that when the [a:] sound was ready to be pronounced was between 2.31 mm and 6.15 mm ; the degree of rotation was between 5.03°and 7.73°; and the time required was between 0.115 and 0.174 seconds.3) The amount of translation from the position of the maximal intercuspal occlusion to that when the mandibular movement stopped with the pronunciation of the [a:]sound was between 3.02 mm and 13.37 mm; the degree of rotation was between 6.42° and 12.81°; and the time required was between 0.194 and 0.326 seconds.4) When compared before and after the [a:] sound was pronounced, the rotation of mandibular condyle tended to occur earlier than translation.5) When comparison was made by dividing the entire movement of mandibular condyle into three periods, the speed of change, both in translation and rotation, tended to be quickest in the second period; as for rotation, the degree of rotation tended to be smaller in the third period compared to that in the first.6) From the result of the present research, it is presu med that the mandibular condyle first moves, featuring rotation, to a comparatively fixed position, and later moves, featuring translation, to the position most appropriate for the pronunciation of the [a:] sound.

9] Cell-free synthesis of noninterstitial (CHL cell) procollagen chains

This chapter presents the methodologies employed to achieve the cell-free synthesis of procollagen chains directed by components of Chinese hamster lung (CHL) cells, which synthesize a noninterstitial collagen. Each of the three cell-free systems described in this chapter offers unique opportunities for investigating the biosynthesis and metabolism of noninterstitial procollagen chains. The CHL S30-catalyzed system is an homologous cell-free system, and as a consequence, any positive or negative translational element(s), i.e., specific tRNA or initiation factor, involved in noninterstitial procollagen chain mRNA expression is presented. The CHL polysome-directed system offers the advantage of allowing the discrimination among transcribed but not translated procollagen chain mRNA species from those that are actively being expressed. The heterologous cell-free protein synthesis system programmed by CHL mRNA offers the opportunity not only to discern positive translational regulatory elements but also to identify factors and enzymes involved in the posttranslational modification(s) and catabolism of a noninterstitial procollagen chain.

The structure and function of the regulatory elements of the Escherichia coli uvrB gene.

The construction and properties of recombinant plasmids carrying the Escherichia coli uvrB gene, including its transcriptional- and translational regulatory elements, is reported. The DNA sequence of the region, which governs the expression of the uvrB gene, has been determined. Within this sequence two non-overlapping DNA segments match the model sequence for Escherichia coli promoters (1). The '-10 regions' and the '-35 regions' of the proposed uvrB promoters are, respectively, 5'TAAAAT (P1), 5'TATAAT (P2) and 5'TTGGCA (P1), 5'GTGATG (P2). The existence and the position of these promoters has been established by elimination of one promoter (P2), using molecular cloning procedures, by length measurements of in vitro synthesized 'run-off' transcripts and by protection of the uvrB regulatory region for S1 nuclease digestion using in vivo made RNA. Potential sites of interaction within the uvrB regulatory region with regulatory proteins, such as the LexA protein (2) and the UvrC protein (3) are discussed.

Sequence of the 5′-end of Strongylocentrotus purpuratus H2b histone mRNA and its location within histone DNA

EXTENSIVE DNA sequence analysis of the cloned histone gene repeat unit from the sea urchin Stronglyocentrotus purpuratus has identified the five histone protein coding sequences1–3 (I.S., unpublished). R-loop and heteroduplex mapping strongly suggest that the sea urchin histone genes contain no intervening sequences4. In addition to the protein coding sequences the corresponding histone mRNAs each contain about 100 extra nucleotides4,5. The location of these additional sequences in relation to the coding sequences is not certain. It is of interest to map the mRNA termini within the known DNA sequence because this information may help to identify transcriptional and translational control elements. Direct nucleotide sequence analysis of RNA can be carried out by enzymatic, base-specific cleavage of end-labelled RNAs6,7. However, 5′-end labelling of mRNAs involves several enzymatic reactions (‘decapping’, dephosphorylation and phosphorylation) that require nuclease-free enzyme preparations8; furthermore, the RNA to be sequenced must be homogeneous. Indirect sequencing analysis of RNA can be obtained from its complementary cDNA. RNA has been sequenced in this way using a chain termination method with dideoxytriphosphates9, synthetic primers and purified mRNA templates10–12. Alternatively, end-labelled restriction fragments have been used to prime synthesis of cDNA on homogeneous 13,14 and partially purified15,16 mRNA templates followed by either chemical17 or enzymatic9 sequencing of the resulting cDNA. Our approach is similar and shows that prior purification of the RNA template is unnecessary. We present here the 5′-terminal sequence of S. purpuratus H2b mRNA, derived from purified H2b mRNA as well as from total polysomal RNA, using the chain termination method9 adapted for RNA sequencing. We also determine its location within the histone DNA sequence.

Force System Structural Synthesis By Using Coupler Curves and Interactive Computer Graphics

An interactive computer graphics program has been developed to structually synthesize force systems which will drive a 4R-4Bar linkage to have a desired dynamic motion time response. The program is also useful for iterative dimensional synthesis. The program contains a coupler curve “boxing” technique to help the designer identify generalized force “characteristic” curve shapes for translational force elements between nonadjacent links. Some general observations about generalized force “characteristic” curve shapes are also presented.

Alternative Tuned Absorbers for Steady State Vibration Control of Tall Structures

Two forms of damped vibration absorbers are evaluated to describe their value in reducing steady state vibration of tall buildings. The first model contains a set of two identical one-degree-of-freedom elements symmetrically mounted in a horizontal plane on either side of the building’s long axis. An alternate model has independent translational and torsional elements mounted at the building’s center. Damping parameters are included for building fundamental bending and torsion modes to evaluate those effects on response. The sensitivity of absorber performance to absorber-building mass ratio μ is of interest to minimize the size of the absorber. Performance of the absorber models was compared based on maximum transmissibility and a quality integral, which is an integrated transmissibility over a frequency spectrum based on amplification at a point on the building top. Small damping in the structure aided absorber performance. Quality is quite insensitive to mass ratio and absorber damping for a building damping ratio in the order of 0.05. Damping is to be reduced in the absorber to improve quality for small absorber-to-building mass ratios when building damping becomes small.

The relationship between function and DNA sequence in an intercistronic regulatory region in phage lambda.

rho factor-mediated transcription termination at the tr1 terminator site of bacteriophage lambda is examined. Mutations affecting the termination event are characterised. These mutations define features of the site which seem to be important to terminator function. In addition, other related transcriptional and translational regulatory elements are defined within the region surrounding the termination site. The potential molecular interactions and structural overlaps of these control signals apparently couple the regulation of the decision between lytic and lysogenic growth patterns by phage lambda.

Simian virus 40-specific ribosome-binding proteins induced by a nondefective adenovirus 2-simian virus 40 hybrid

We have studied the intracellular distribution of the two simian virus 40-specific proteins, with apparent molecular weights of 56,000 and 42,000, detectable in human KB cells infected by a nondefective adenovirus 2-simian virus 40 hybrid, Ad2+ND2. After a 20-min pulse of [35S]methionine, about two-thirds of the newly synthesized 56K protein and one-third of the 42K protein were found localized on the plasma membrane. The remainder of each protein was found in the cytoplasm, whereas the nuclear fraction was virtually free of either component. A significant portion of both proteins present in the cytoplasmic fraction was complexed to the 40S ribosomal subunits and was not removed by treatment with 0.5 M KCl. Moreover, the portion that was found free in the cytoplasm could bind preferentially and quantitatively to purified 40S ribosomes in vitro, leading us to propose that these simian virus 40 proteins may act as translational control elements in cells.

Translation of Xenopus liver messenger RNA in Xenopus oocytes: Vitellogenin synthesis and conversion to yolk platelet proteins

Xenopus liver vitellogenin and albumin mRNAs injected into Xenopus oocytes are correctly translated, as shown by specific immunoprecipitation and co-electrophoresis with purified Xenopus vitellogenin (molecular weight 210,000 daltons) and albumin (molecular weight 72,000 daltons). Vitellogenin made in oocytes under the direction of injected liver mRNA is unstable compared to other proteins made on injected messengers (such as albumin and globin) and endogenous oocyte proteins (including actin), the half-life of newly made vitellogenin being about 8 hr. Pulse-chase experiments with 35S-methionine show vitellogenin to be a precursor to yolk platelet lipovitellin (molecular weight 120,000 daltons), while 3H-serine labeling demonstrates conversion to phosvitin (molecular weight 34,000 daltons). In contrast, injected 3H-serine 35S-methionine-labeled Xenopus vitellogenin protein is not converted to yolk platelet proteins and is degraded rather slowly (half-life, 23–29 hr). Phosphorylation of serine residues in phosvitin can be detected in oocytes injected with 32PO 4 or γ- 32P-ATP; thus exogenously derived yolk platelet protein is further modified, or turned over, once it is within the oocyte. Moreover, vitellogenin made in oocytes programmed with liver mRNA is phosphorylated. Thus phosphorylation, assembly into yolk platelets, and cleavage are events that do not require vitellogenin supplied by the normal pathways involved in yolk formation (synthesis and post-translational modification in the liver, transport in the serum, and follicle cell-dependent pinocytosis). Vitellogenin mRNA sediments at about 29S in a sucrose-SDS gradient, while albumin messenger peaks at 16S; both species contain poly(A). These liver mRNAs are functionally stable in oocytes for at least 5 days. Vitellogenin-forming activity, relative to albumin, actin, or total endogenous activity, increases with time, and the final rate of 2–2.5 times the initial rate is only reached 3 days after injection. The potentiation effect probably stems from an increase in the efficiency of translation of vitellogenin mRNA. The availability of homologous mRNAs now permits injected messenger to be used as a valid probe of oocyte function: the biological activity of mRNA from a non-ovarian Xenopus tissue proves that some at least of the translational systems within the Xenopus oocyte are not cell type-specific. Moreover, the whole cell system is eminently suitable for assaying putative translational (and possibly transcriptional) control elements from frog liver.

Host factor for coliphage Qbeta RNA replication is present in Pseudomonas putida.

Host Factor (HF)1, is a 12000 molecular weight polypeptide that is found in uninfected Escherichia coli and is required as a hexamer along with Qbeta replicase for in vitro replication of Qbeta phage RNA. It has recently been found to be associated with ribosomes and to bind tightly to poly(A). We report here the identification and purification of HF from Pseudomonas putida. HF can be detected in crude extracts by both functional activity in the Qbeta RNA replication assay and by immunodiffusion with antibody made against E. coli HF. HF from E. coli and P. putida chromatograph similarly on DEAE-cellulose and phosphocellulose. They have similar but not identical molecular weights as judged by SES-polyacrylamide gel electrophoresis. Like E. coli HF, P. putida HF was found to be associated with ribosomes and to bind tightly to poly(A). Furthermore, the pure protein from P. putida has full funcitonal activity in the in vitro Qbeta RNA replication assay. The findings that HF has been conserved during evolution, is associated with ribosomes, and binds poly(A), suggest that HF may be an important translational element in uninfected cells and that its role involves an interaction with RNA.