Functional Polypeptides Research Articles

Evolutionary changes of nucleotide sequences of papova viruses BKV and SV40: they are possibly hybrids.

Complete nucleotide sequences were compared between papova viruses BKV and SV40 and the degrees of sequence divergences were compared between structurally and/or functionally different segments or genes in details. It was shown that the rate of synonymous substitution is not only very high but also approximately uniform among different genes in these viruses as in eukaryotic genes examined to date. While all the non-coding regions including the intron showed marked sequence preservation which is in sharp contrasted with the case of eukaryotic genes where the large bulk of non-coding regions evolve at a rate as rapidly as that of synonymous substitution. It is remarkable that a long continuous stretch of sequence including the putative VPX gene and a 5' half of VP2 gene showed strong homology between BKV and SV40. A close examination of the pattern of base substitutions revealed that this unusual homology was derived by recombination between the two viruses during their evolution. On the basis of the pattern of base substitutions and the bias in code word utilization, we also showed that the putative VPX gene actually could code for a functional polypeptide. In papova viruses, the 3' terminal sequence of VP2/3 gene overlaps with the 5' terminal sequence of VPI gene. The pattern of base substitutions in the overlapping segment was examined in detail in comparison with those in the non-overlapping portions of VP2/3 and VP1 genes. It was shown that the evolutionary mode of the overlapping genes is in good agreement with our previous prediction.

The structure of the human zeta-globin gene and a closely linked, nearly identical pseudogene

DNA sequencing studies indicate that only one of two closely linked human embryonic α-like globin genes, zeta (ζ), encodes a functional polypeptide. The other is a pseudogene (ψζ) that differs by only 3 bp in the protein coding sequence, one of which converts the codon for amino acid 6 into a chain termination codon. Both ζ-globin genes differ from all other α-like genes thus far reported in that they contain large introns consisting, in part, of simple repeat sequences. Intron 1 of each gene contains a variation of the repeat sequence ACAGTGGGGAGGGG, while intron 2 contains the repeat sequence CGGGG. Comparison of the human ζ- and α-globin gene sequences reveals that the embryonic and adult α-like genes began to diverge from each other relatively early in vertebrate evolution (400 million years ago). In contrast, the β-like embryonic globin gene, epsilon (ϵ), is the product of a much more recent evolutionary event (200 million years ago). Thus, even though the temporal and quantitative expression of ζ- and ϵ-globin genes must be coordinately controlled during development, their evolutionary histories are clearly distinct.

Monosaccharide transporter of the human erythrocyte. Characterization of an improved preparation.

The human erythrocyte monosaccharide transporter has been purified through the use of the dialyzable detergent octyl glucoside. It was found that the transporter denatures in the detergent and that the rate of this process could be reduced by increasing the ratio of phospholipid to detergent. The transporter was obtained in higher yield and with a higher specific activity for cytochalasin B binding than has been previously reported. Scatchard plot analysis of cytochalasin B binding to the reconstituted preparations gave a dissociation constant of 1.5 X 10(-7) M, and there were found to be 15.3 nmol of sites/mg of protein. On the basis of a value of 46 000 for the molecular weight of the polypeptide, this specific activity corresponds to 0.70 site/polypeptide chain; and there are reasons to believe that the value of the stoichiometry may be one site per functional transporter polypeptide. The complete amino acid composition and the N- and C-terminal residues of the transporter have been determined. Both the intact transporter and transporter that had been partially depleted of carbohydrate by treatment with endo-beta-galactosidase were found to migrate anomalously upon sodium dodecyl sulfate--polyacrylamide gel electrophoresis, relative to the behavior of standard proteins.

THE EFFECT OF A MUTATION CAUSING ALCOHOL DEHYDROGENASE DEFICIENCY ON FLOODING TOLERANCE IN BARLEY

Summary Adh1‐M9 mutant barley plants lack a functional ADH1 gene product. The effect of this deficiency in alcohol dehydrogenase (ADH) activity on flooding tolerance is investigated by comparing the behaviour of mutant and wild‐type plants in flooded conditions. It is concluded that there is a specific requirement for a functional ADH1 polypeptide in the immersed seed but not at later stages of development. The significance of these findings is discussed with respect to current theories of flooding tolerance.

Procedure for production of hybrid genes and proteins and its use in assessing significance of amino acid differences in homologous tryptophan synthetase alpha polypeptides.

Hybrid tryptophan synthetase alpha and beta polypeptides were produced by genetic recombination between the trpB--trpA regions of Escherichia coli and Salmonella typhimurium contained on compatible, multicopy plasmids. Intragenic recombination was decreased but still evident in recA cells. Genetic exchange occurred at many sites within trpA, but every recombinant gene produced a functional alpha polypeptide despite many amino acid differences from one or the other of the parental polypeptides. The five hybrid tryptophan synthetase alpha subunits examined resembled the parental polypeptides in catalytic function but differed in thermostability. The stability differences suggest that, as amino acid changes occurred in these proteins during the course of evolution, subsequent changes were limited to those that would allow retention of a desired protein conformation.

Rapidly evolving mouse alpha-globin-related pseudo gene and its evolutionary history.

The nucleotide sequences of a mouse pseudo alpha-globin gene and two adult alpha-globin genes from mouse and rabbit were compared. A close examination of sequence differences among the three genes revealed that the mouse pseudo alpha-globin gene was derived from one of the mouse alpha-globin genes about 24 million years ago by gene duplication and lost its original function, presumably due to loss of both the intervening sequences or frameshift mutations that prevented production of a functional globin polypeptide, and eventually became an inactive gene about 17 million years ago. After this event, this gene evolved at a very high rate, approximately 1.9 times the rate of synonymous codon change in productive genes. This suggests the presence of functional constraints against synonymous codon changes in normally functioning genes and also suggests that the "nucleotide arrangements" serve almost no important functions beside protein coding ability in the greater part of this gene, except for a very limited number of nucleotides. A continuous stretch of the pseudo alpha-globin gene consisting of a third exon and a 5' half of the 3' noncoding region shows marked sequence homology to the alpha-globin gene, suggesting transfer from one of the globin or globin-like genes by recombination very recently.

Isolation and characterization of a heparin-binding domain of cellular fibronectin.

A protease-resistant functional polypeptide domain of fibronectin that contains a heparin-binding site has been purified from chick cellular fibronectin following pronase digestion, heparin-Sepharose affinity chromatography, and molecular sieve chromatography on Sephacryl S-200. This polypeptide migrates as a single band of apparent Mr = 50,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with or without reduction by dithiothreitol. It has a Stokes radius of 35 to 37 A and a calculated frictional ratio of 1.4 to 1.5. Its amino acid composition is unusual in that cysteine and methionine constitute only 1 and 2 mol/mol of the polypeptide, respectively. The NH2-terminal amino acid sequence is Glu-Asp-Asp. In contrast to intact fibronectin, the heparin-binding polypeptide contains virtually no neutral sugars. This 50,000-dalton domain is inactive in four bioassays for intact fibronectin, including hemagglutination, spreading of cells on tissue culture plastic, restoration of alignment of tumor cells, and attachment of cells to collagen. Moreover, excess amounts of this polypeptide do not inhibit the activity of intact fibronectin in all of these assays; these results suggest that the heparin-binding domain does not contain a cell surface-binding site. This domain of fibronectin appears to be a unique region free of carbohydrate that is a globular, protease-resistant functional site.

The nucleotide sequence of a rabbit beta-globin pseudogene.

We report the nucleotide sequence of a rabbit beta-globin pseudogene, psi beta 2. A comparison the psi beta 2 sequence with that of the rabbit adult beta-globin gene, beta 1, reveals the presence of frameshift mutations and premature termination codons in the protein coding sequence which render pse beta 2 unable to encode a functional beta-globin polypeptide. psi beta 2 contains two intervening sequences at the same locations in the globin protein coding sequence as beta 1 and all other sequenced beta-globin genes. An examination of the DNA sequences at the intron/exon junctions suggests that a putative pse beta 2 precursor mRNA could not be spliced normally. We compare the flanking and noncoding sequences of pse beta 2 and beta 1 and discuss the evolutionary relationship between these two genes.

Models for decay of Escherichia coli lac messenger RNA and evidence for inactivating cleavages between its messages

The size distributions of decaying polycistronic Escherichia coli lac messenger RNA have been followed on polyacrylamide gels. At the same time, equations have been derived that generate the theoretical size distributions of decaying macromolecules for different mechanisms of degradation. Using observed values of lac mRNA metabolism, it was possible to reproduce the in vivo patterns with a model in which cleavage occurs at the start of each of the three messages † † Abbreviations used: message, mRNA coding for a single, functional polypeptide (a polycistronic mRNA carries several messages); βGase, β-galactosidase (EC 3.2.1.23) coded by z gene; TA or transacetylase, galactoside acetyltransferase (EC 2.3.1.18) from the a gene; fmet-tRNA, formylmethionyl-tRNA; IF, initiation factor(s). and is followed by a net 5′ to 3′ wave of mass loss. Other models of degradation could not generate the observed in vivo patterns. These alternative mechanisms include: (1) the same number of primary cleavage sites (three) but at different positions on the full-length molecule; (2) an exclusive 5′ to 3′ directional degradation from the start of the lac mRNA (no cleavages); or (3) the presence of many internal targets. Further support for primary cleavage at the start of messages came from the observed accumulation of the intact z mRNA released by cleavage at the z y boundary and from the predictable effects of specific deletions on the resultant size distributions. The significance of these cleavages has been assessed; they could be necessary “processing” events or, conversely, inactivate the message for translation. Full-length molecules as well as cleavage fragments have been fractionated by successive sucrose gradient centrifugation and tested for their capacity to form translation initiation complexes in vitro. Full-length lac RNA could form such complexes at one or more of its three ribosome-loading sites, whereas the y or a message fragments were inactive. These results suggest that a cleavage at or near the start of a message inactivates it.

Rubella virus: Intracellular polypeptide synthesis

Rubella virus (RV) grown in RK13 cells induces a range of polypeptides ranging in molecular weights from 109K to 28K. As RV does not inhibit host metabolism, detection of these requires the use of elevated concentrations of NaCL in the labelling medium to selectively inhibit cellular translation. Several of the viral polypeptides, including p109, p92, and p72 are transient high molecular weight species which may represent precursors of functional viral polypeptides. Only five or six of the polypeptides are synthesized in large amounts at late stages of infection and these probably include the structural components.

Freeze-fracture studies on barley plastid membranes. VI. Location of the P700-chlorophyll a-protein 1.

The photosystem I mutant of barley, viridis-zb63, which totally lacks the P700-chlorophyll a-protein 1 was characterised by rotary shadowed, freeze-fracture electron microscopy. Objective measurements were made of particle density and size distribution for all four fracture faces, and compared with values for wild-type. A highly significant reduction in the size of the PFu particles was found, which could be attributed to the loss of a population of large particles from the mutant PFu face. A corresponding loss of EFu pits was also observed. It is concluded that the photosystem I reaction centre and two or three ancillary polypeptides are located in the large PFu particles which account for two-thirds of the total, and that these particles span the membrane. Since no differences were seen on the PFs and EFs faces, there was no evidence for the localisation of any photosystem I in appressed granal membranes, supporting the concept of extreme lateral heterogeneity. A model is presented of the localisation of different functional polypeptide units to different freeze-fracture particles within the membrane. A peculiar feature of viridis-zb63 thylakoids was the presence of EFs particle arrays in vivo.

On pre-messenger RNA and transcriptons a review

From the present review integrating old and new data emerge a few principles of gene expression in eukaryotes, and an infinite variety of possible mechanistic details generating the overal pattern. The few principles, most of which are not fundamentally new, may thus be summarized. 1) The eukaryotic genome is subdivided into transcriptional units: into transcriptons which are subject to individual activation controlled at DNA level. 2) Viral genomes may contain one or a few transcriptons, while cells of multicellular organisms contain from 3 x 10(3) in diptera up to an estimated 2 x 10(5) in birds and mammals. 3) Transcriptons may include one or several coding sequences. 4) Transcriptons vary considerably in size: in mammals and birds their size spectrum falls into the 2,000 to 20,000 bp range. 5) Units of coding information constituting one message (genes) and, possibly, units of regulative information are frequently broken up and stored within the transcripton in sub-genic blocks (of so far unknown significance) in general located at a certain distance from the 5' and 3' transcript terminals which are determined by the promotor and terminator signals. 6) The gene, in its specific definition as the functional unit underlying the phenotype, is in general constituted posttranscriptionally by the processing mechanisms from the mosaic of its genomic subunits in the transcripton; segments of coding, service and regulative sequences are recombined within themselves and with each other, polygenic transcripts separate into their unit messages. 7) Activated transcriptons produce pre-mRNA; these primary transcripts are colinear with the DNA of the transcriptional unit. 8) Primary pre-mRNA is processed into secondary pre-mRNA's by extragenic cleavage and intragenic ("splicing") processing, giving rise stepwise to functional mRNA. During this process chemical modifications as methylation, 5'-terminal capping and 3'-terminal polyadenylation take place. 9) Translation yields either potentially functional polypeptides or polycistronic polyproteins subject to further processing. 10) Processing is a regulated process; it involves many of the possible phases and mechanisms of post-transcriptional regulation (cf. 39, 40).

Studies of Ultrafine Metal Particles Prepared by the Addition of Watersoluble Protein

Ultrafine cobalt and cobalt alloy particles have been prepared by spontaneous autocatalytic reduction.The influence of parameters such as protein structure, magnetic field applied for reduction and reduction reagent on particle growth was investigated. Ultrafine cobalt particles with the size ranging from 60 to 700Å can be easily obtained by using various kinds of proteins and thier particle sizes markedly depend on the structure of proteins used. The suggested explanation of protein effect is that the nuclei of cobalt particle are formed on each functional polypeptide group of the macromolecular protein. The strength of magnetic field, which is applied for reduction, should be more than twice as large coercive force of cobalt and cobalt alloy partticles. X-ray diffraction studies were carried out on the samples obtained by the reduction of hydrazine and hypophosphite. Cobalt particles obtained by the hydrazine reduction have almost fee crystal structure, and not in those containing phosphorous. However, in the case of hypophosphite reduction, the particles are crytalline hcp. This behavior may be explained by the increase of Co-P components.

Expression in Escherichia coli of a Chemically Synthesized Gene for the Hormone Somatostatin

A gene for somatostatin, a mammalian peptide (14 amino acid residues) hormone, was synthesized by chemical methods. This gene was fused to the Escherichia coli beta-galactosidase gene on the plasmid pBR322. Transformation of E. coli with the chimeric plasmid DNA led to the synthesis of a polypeptide including the sequence of amino acids corresponding to somatostatin. In vitro, active somatostatin was specifically cleaved from the large chimeric protein by treatment with cyanogen bromide. This represents the first synthesis of a functional polypeptide product from a gene of chemically synthesized origin.

Macromolecular synthesis in cells infected by frog virus 3. VII. Transcriptional and post-transcriptional regulation of virus gene expression.

We have used improved techniques for separating individual species of RNA and protein to study the mechanisms that control gene expression by frog virus 3, a eucaryotic DNA virus. Forty-seven species of viral RNA and 35 viral polypeptide species were resolved by polyacrylamide gel electrophoresis. The relative molar ratios of virus-specific polypeptides synthesized at various times after infection were determined by computer planimetry and were compared with the molar ratios of appropriate-sized viral RNAs to code for each polypeptide. Viral polypeptides were classified according to the time during the growth cycle at which their maximal rate of synthesis occurred - early, 2 to 2.5 h; intermediate, 4 to 4.5 h; and late, 6 to 6.5 h. The viral RNAs, which were assumed to be mRNA's, could not be classified according to time of maximum synthesis; once their synthesis had begun, most of the RNAs continued to be synthesized at the same or higher rates. However, only 10 of the 47 viral RNA bands were plainly visible after electrophoresis of extracts from cells labeled from 1 to 1.5 h after infection; these 10 RNAs were designated "early" RNA. The early pattern of both RNA and polypeptide synthesis was maintained for at least 6 h in the presence of the amino acid analog fluorophenylalanine, which indicates that a functional viral polypeptide was required for "late" transcription and translation. The presumptive mRNA's for late polypeptides did not appear until 2 h after infection, but two of these "late" RNAs became the major products of transcription by 4 h into the infectious cycle. In contrast to the declining rate of synthesis of the early proteins, corresponding early RNA species continued to be synthesized at the same or higher rates throughout the replicative cycle. Although the synthesis of late virus-specific proteins appeared to be regulated at the level of transcription, our results suggest that the synthesis of both early and intermediate proteins was regulated at the post-transcriptional level.

Transcription and translation initiation frequencies of the Escherichia coli lac operon

We have calculated the number of RNA polymerase molecules transcribing the induced lac operon of Escherichia coli as well as the distance between ribosomes on the proximal z and on the distal a messages † † Abberviations used: message, mRNA coding for a single, functional polypeptide (a polycistronic mRNA carries several messages); βGase, β-galactosidase (EC 3.2.1.23) coded from z gene; GATase, galactoside acetyltransferase (EC 2.3.18) coded from the a gene. . These values were derived from: (a) rates of induced enzyme synthesis corrected by known turnover numbers to give numbers of enzyme monomers produced per cell per second; (b) the mass of each message derived either directly by hybridization of long-labeled RNA to specific DNA or from the rates of synthesis and decay obtained by hybridization of pulse-labeled RNA; the latter gives about 13 times better resolution; (c) conversion of message mass into a number of funtioning 3′ ends (producing finished polypeptides); size analyses indicate that the z message probably decays by a net directional degradation in the 5′ to 3′ direction. From this the fraction of completed molecules that are full-length and the total number with functional 3′ ends can be derived. (d) Rates of ribosome movement. Calculated values follow. (1) With a 3.3-second interval between transcription initiations, there are 38 molecules of RNA polymerase on z DNA per cell; with 1.7 copies of lac DNA, this gives 23 on each z cistron and five or six on the y and on the a cistrons. This corresponds to a spacing of about 135 nucleotides between polymerases, which is similar to that on the ribosomal cistrons which have only 1.7 seconds separating initiations. (2) There are 20 molecules of β-galactosidase monomer produced per cell per second from 1.1 × 10 −4 pg of z RNA. 38% of the message molecules are nascent and not producing enzyme. Half of the completed molecules and 70% of their mass are intact z messages to give 62 molecules of z message containing functional 3′ ends/cell. Ribosomes load onto these messages at 3.2-second intervals to give a ribosomal spacing of 110 nucleotides. An average of 40 ribosomes translate each z message with half of the message molecules translated by more than 28 ribosomes and half by less. (3) Only 2.4 molecules of galactoside acetyltransferase monomer are produced per cell per second from 1.8 × 10 −5 pg of a RNA. Only 20% of these molecules are nascent and 80% of the completed ones are intact; this gives 40 molecules of functioning a messages per cell. Ribosomes load to a message at 16-second intervals to give a spacing of 580 nucleotides. These results show that the frequencies of translation initiations can differ for different messages. The faster decay of a compared to z message is consistent with a model in which a ribosome can protect a vulnerable site near the start of a message from inactivation; messages that load less frequently decay faster. The combined net effect of these causally related processes could account for natural polarity (Zabin & Fowler, 1970), i.e. there is a fourfold lower production of enzyme from the a than from the z gene.

Structure and properties of a hybrid tryptophan synthetase of alpha chain produced by genetic exchange between Escherichia coli and Salmonella typhimurium.

Genetic exchange between the structural genes for the alpha chain of tryptophan synthetase [tryptophan synthase; L-serine hydro-lyase (adding indoleglycerol-phosphate), EC 4.2.1.20] of E. coli and S. typhimurium yielded recombinant genes that specified functional hybrid polypeptides. The alpha chains produced by three recombinants appeared to be identical but differed from those of E. coli and S. typhimurium by at least 27 and 8 amino acid residues, respectively. In vivo and in vitro tests of enzyme function suggest that the hybrid alpha chains are near-equivalent to their fully active parental proteins.

Cell killing by viruses: III. The interferon system and inhibition of cell killing by vesicular stomatitis virus

The dose response of cell killing particle (CKP) activity of vesicular stomatitis virus (VSV) in GMK Vero cells treated with interferon or mouse L cells treated with poly(rI)·poly(rC) was determined from single-cell survival curves, and compared with dose-response curves for infectivity and viral transcript accumulation. “Pulse-infection” with temperature-sensitive (ts) mutants of VSV was used to confine formation of putative cell killing factor or its precursors to the time equivalent of a single cycle of growth. Cells which through interferon or poly(rI)·poly(rC) action survived a pulse-infection with ts-mutant virus at permissive temperature produced colonies normal in both appearance and growth rate. Cells treated with interferon or poly(rI)·poly(rC) and challenged with VSV generated a family of survival curves for CKP activity whose slopes decreased as the dose of the interferon-interference inducer increased. These survival curves revealed that the capacity to express CKP activity or accumulate transcripts in interferon-treated Vero cells were both lost initially at an exponential rate about four to five times slower than that observed for the loss of plaque or yield reducing capacity, demonstrating that it required four to five times more interferon to prevent cell killing or transcript accumulation by VSV than was needed to reduce infectivity by an equivalent amount. A marked tailing of the survival curves was observed as the interferon dose was increased, resulting in surviving activity for CKP and transcript accumulation far in excess of that expected by extrapolation from low-dose regions of the curve. These results are discussed in light of our previous hypothesis which stated that viral transcription was a requisite, though insufficient reaction to produce cell killing by VSV and that integrity of about one-fifth of the viral genome sufficed to provide these transcripts (Marcus, P. I., and Sekellick, M. J. (1974). Virology 57, 321–338; (1975). Virology 63, 176–190). We extend this hypothesis and propose that the lethal action of VSV on cells requires: (i) functional virion transcriptase, (ii) transcription of a special one-fifth of the genome, most likely that coding for proteins N and NS, (iii) translation of these transcripts into minimally functional polypeptides which lead to (iv) formation and subsequent action by a putative cell killing factor.

Regulation of herpesvirus macromolecular synthesis: sequential transition of polypeptide synthesis requires functional viral polypeptides.

It was previously shown that virus-specific polypeptides made in HEp-2 cells infected with herpes simplex 1 form three groups designated alpha, beta, and gamma whose synthesis is coordinately regulated and sequentially ordered. This report shows that one or more functional alpha polypeptides are necessary to turn on the synthesis of beta and gamma groups, and conversely, one or more polypeptides in the latter groups turn off the synthesis of alpha polypeptides. Specifically, infected cells maintained in medium containing either canavanine, an analogue of arginine, or azetidine-2-carboxylic acid an analogue of proline and hydroxyproline, synthesized alpha polypeptide at rates comparable to maximal rates in untreated infected cells but did not undergo the normal transition to beta and gamma polypeptide synthesis. The transition to gamma polypeptide synthesis and shut-off of synthesis of earlier polypeptide groups proceeded normally if addition of canavanine was delayed until at least 4-5 hr after infection. Addition of canavanine after the onset of beta and gamma polypeptide synthesis, i.e., between 2 and 3.5 hr after infection, resulted in sustained, simultaneous synthesis of all three polypeptide groups, a phenomenon not seen in untreated infected cells. Canavanine-treated infected cells, synthesizing alpha polypeptides, recovered the capacity to make beta and gamma polypeptides after removal of the analogue, but only after a 1-to 2-hr delay compared with infected untreated cells. The data indicate that the on and off controls inherent in the cascade regulation of viral polypeptide synthesis are mediated by one or more polypeptides in each group at transcriptional or post-transcriptional levels.