Recombination Between Guanidine-resistant and Dextran Sulfate-resistant Mutants of Type 1 Poliovirus

Mixed infection of monkey kidney cells with two mutants of the LSc2ab strain of poliovirus, one resistant to guanidine and the other resistant to both dextran sulfate and 2-(alpha-hydroxybenzyl)-benzimidazole (HBB), yielded progeny in which the number of gua(r)dex(r) particles exceeded by a factor of 7 to 10 the expected number of similar particles occurring through spontaneous mutation; recombination would explain the fairly high excess of doubly mutant particles that was obtained. Scoring of HBB resistance in 50 gua(r)dex(r) clones suggested that, during recombination, resistance to dextran sulfate is not associated with HBB resistance.     

Chlorophyll-Protein Complexes from Euglena gracilis and Mutants Deficient in Chlorophyll b: II. Polypeptide Composition

Chlorophyll-protein complexes (CPs) obtained from thylakoids of Euglena gracilis Klebs var bacillaris Cori contain the following polypeptides (listed in parentheses in order of prominence after Coomassie R-250 staining of polyacrylamide gels): CP Ia (66, 18, 22, 22.5, 27.5, 21, 28, 24, 25.5, and 26 kilodaltons [kD]); CP I (66 kD); CPx (41 kD); LHCP₂ (an oligomer of LHCP) (26.5, 28, and 26 kD); CPy (27 and 19 kD); CPa (54 kD); and LHCP (26.5, 28, and 26 kD). Mutants of bacillaris low in chlorophyll b (Gr₁BSL, G₁BU, and O₄BSL; Chl a/b [mol/mol] = 50-100) which lack CP Ia, LHCP₂, and LHCP also lack or are deficient in polypeptides associated with these complexes in wild-type cells. Mutants G₁ and O₄, which also lack CPy, lack the CPy-associated polypeptides found in wild-type and Gr₁. Using an antiserum which was elicited by and reacts strongly and selectively with the SDS-treated major polypeptide (26.5 kD) of the LHCP complexes of wild-type, this polypeptide is undetectable in the mutants (0.25% of the level in wild-type on a cell basis); the antiserum does not react with the SDS-treated 28 kD polypeptide of the Euglena LHCP complexes and cross-reacts only very weakly with components in SDS-treated cells of Chlamydomonas reinhardtii Dangeard and chloroplasts of Spinacia oleracea L. cv Winter Bloomsdale. Rates of photosynthesis of the wild-type and mutant cells of Euglena are approximately equal on a cell basis when measured at light saturation, consistent with the selective loss of major antenna components but not CP I or CPa from the mutants.     

Spectral Properties and Composition of Reaction Center and Ancillary Polypeptide Complexes of Photosystem II Deficient Mutants of Synechocystis 6803

The polypeptide composition and spectral properties of three photosystem II (PSII) deficient mutants of the cyanobacterium Synechocystis 6803 have been determined. The levels of the 43 and 47 kilodalton chlorophyll-binding proteins and the reaction center component D2 are affected differently in each mutant; the 33 kD polypeptide of the oxygen-evolving complex is found at wild-type levels in all three. The 43 and 47 kilodalton proteins are implicated as important elements in the assembly and/or stability of the PSII reaction center, although the loss of one of these polypeptides does not lead to the loss of all PSII proteins. Low temperature fluorescence emission spectra of wild-type cells reveal chlorophyll-attributable peaks at 687 (PSII), 696 (PSII), and 725 (photosystem I) nanometers. All three mutants retain the 725 nanometer fluorescence but lack the 696 nanometer peak. This suggests that the latter fluorescence arises from PSII reaction center chlorophyll or results from interactions among functional PSII components in vivo. Cells that contain the 43 kilodalton and lack the 47 kilodalton protein, retain the 687 fluorescence; furthermore, in as much as this fluorescence is absent from cells without the 43 kilodalton protein, the 687 nanometer peak is judged to emanate from the 43 kilodalton chlorophyll-protein. A new peak, probably previously obscured, is revealed at 691 nanometers in cells that retain the 47 kilodalton protein but lack the 43 kilodalton polypeptide, suggesting that emission near 691 nanometers can be attributed to the 47 kilodalton polypeptide. Membrane-bound phycobilisomes are retained in these cells as is coupled-energy transfer between phycocyanin and allophycocyanin. Energy transfer to photosystem I by way of phycocyanin excitation proceeds as in wild-type cells despite the absence of certain PSII components.     

Poliovirus Polyuridylic Acid Polymerase and RNA Replicase Have the Same Viral Polypeptide

A poliovirus-specific polyuridylic acid [poly(U)] polymerase that copies a polyadenylic acid template complexed to an oligouridylic acid primer was isolated from the membrane fraction of infected HeLa cells and was found to sediment at 4 to 5S on a linear 5 to 20% glycerol gradient. When the poly(U) polymerase was isolated from cells labeled with [(35)S]methionine and was analyzed by glycerol gradient centrifugation and polyacrylamide gel electrophoresis, the position of only one viral protein was found to correlate with the location of enzyme activity. This protein had an apparent molecular weight of 62,500 based on its electrophoretic mobility relative to that of several molecular weight standards and was designated p63. When the poly(U) polymerase was isolated from the soluble fraction of a cytoplasmic extract, the activity was found to sediment at about 7S. In this case, however, both p63 and NCVP2 (77,000-dalton precursor of p63) cosedimented with the 7S activity peak. When the 7S polymerase activity was purified by phosphocellulose chromatography, both p63 and NCVP2 were found to co-chromatograph with poly(U) polymerase activity. The poliovirus replicase complexed with its endogenous RNA template was isolated from infected cells labeled with [(35)S]methionine and was centrifuged through a linear 15 to 30% glycerol gradient. The major viral polypeptide component in a 26S peak of replicase activity was p63, but small amounts of other poliovirus proteins were also present. When the replicase-template complex was treated with RNase T1 before centrifugation, a single peak of activity was found that sedimented at 20S and contained only labeled p63. Thus, p63 was found to be the only viral polypeptide in the replicase bound to its endogenous RNA template, and appears to be active as a poly(U) polymerase either as a monomer protein or as a 7S complex.     

Poliovirus Mutants at Histidine 195 of VP2 Do Not Cleave VP0 into VP2 and VP4

The final stage of poliovirus assembly is characterized by a cleavage of the capsid precursor protein VP0 into VP2 and VP4. This cleavage is thought to be autocatalytic and dependent on RNA encapsidation. Analysis of the poliovirus empty capsid structure has led to a mechanistic model for VP0 cleavage involving a conserved histidine residue that is present in the surrounding environment of the VP0 cleavage site. Histidine 195 of VP2 (2195H) is hypothesized to activate local water molecules, thus initiating a nucleophilic attack at the scissile bond. To test this hypothesis, 2195H mutants were constructed and their phenotypes were characterized. Consistent with the requirement of VP0 cleavage for poliovirus infectivity, all 2195H mutants were nonviable upon introduction of the mutant genomes into HeLa cells. Replacement of 2195H with threonine or arginine resulted in the assembly of a highly unstable 150S virus particle. Further analyses showed that these particles contain genomic RNA and uncleaved VP0, criteria associated with the provirion assembly intermediate. These data support the involvement of 2195H in mediating VP0 cleavage during the final stages of virus assembly.     

Characterization of Some Poliovirus Temperature-Sensitive Mutants and Poliovirus-Related Particle Formation Under Nonpermissive Conditions

Investigation of 15 poliovirus temperature-sensitive (ts) mutants by using physiological tests [formation of virus-specific antigen and ribonucleic acid (RNA) under nonpermissive conditions] permitted us to divide them into three groups. From each group, one mutant was selected (ts 2, 5, 11), and a comparative study of poliovirus-related particle (5, 10, 73, and 150S) formation under permissive (36 C) and nonpermissive (40 C) conditions was carried out. The ts 2 and ts 11 are mutants with greatly reduced RNA synthesis which at 40 C produce particles with a sedimentation constant of 5S, and the ts 5 (RNA(+)) mutant produces both 5 and 10S particles. The relationship between different temperature-sensitive defects in the mutants is discussed. The results obtained indicate a possible role of 5S protein structures in morphogenesis of poliovirus.     

重叠PCR 法构建XLRS1 三种不同类型突变体

目的:构建X连锁视网膜劈裂(XLRS)三种不同类型突变体(点突变、缺失突变、插入突变),以便进一步构建不同类型的突变细胞模型。方法:根据NCBI gene数据库中XLRS1基因的CDS设计三对不同类型的引物,使用重叠PCR法获得突变片段,然后克隆入载体pLJM1-EGFP。结果:经测序验证,成功构建了三种不同类型突变体。结论:使用重叠PCR法构建三种不同类型突变体,为进一步研究XLRS1不同类型突变的致病机制奠定了基础。     

诱变选育法夫酵母耐热突变株及基因组变异检测

法夫酵母（Xanthophyllomyces dendrorhous）发酵生产虾青素中存在发酵温度低的主要问题。为了获得耐中温的突变菌株,应用化学诱变,筛选能在25 ℃下生产虾青素且稳定遗传的耐热突变菌株,进一步通过基因组重测序,对突变菌株进行变异检测。筛选出一株法夫酵母突变株YB25,其生长温度为25 ℃,经五代培养后虾青素产量无显著差异。在培养120 h后,YB25的虾青素产量达到237.19 μg/g,比野生型提高29%。通过比较基因组分析,获得了YB25的遗传图谱和遗传变异结果,检测到单核苷酸多态性位点数626个,小片段的插入和缺失序列数184个,结构变异数703个,基因组片段的拷贝数变异293个。通过对变异基因分析发现,脂代谢相关基因及虾青素合成酶基因的变异可能是突变菌株中温高产虾青素的原因。     

Relation between Aggregation Behavior of Glutenin and Its Polypeptide Composition

It was found in the previous studies that the quality of flour is closely related to the aggregation behavior of gluten separated from it, and that difference of aggregation behavior of gluten was mainly due to the nature of glutenin contained in it. In the present paper, aggregation behaviors of glutenins were investigated in relation to their polypeptide compositions with ten kinds of flours. Component polypeptides of glutenin were fractionated into three fractions, FI, FII and FIII, by gel filtration, and their polypeptide compositions and aggregation behaviors were investigated. The ratio of the three fractions was different among the ten glutenins, and a correlation was found between τ₁₀/C values, a parameter for aggregation reaction, of glutenins and their FI and FII contents. τ₁₀/C decreased with increasing content of FI and with decreasing content of FII. Therefore, the aggregation behavior of gluten depends at least partly on the contents of FI and FII in glutenin.     

Pigment Composition and Photosynthetic Activity of Pea Chlorophyll Mutants

Pea chlorophyll mutants chlorotica 2004 and 2014 have been studied. The mutants differ from the initial form (pea cultivar Torsdag) in stem and leaf color (light green in the mutant 2004 and yellow-green in the mutant 2014), relative chlorophyll content (approximately 80 and 50%, respectively), and the composition of carotenoids: the mutant 2004 contains a significantly smaller amount of carotene but accumulates more lutein and violaxanthin; in the mutant 2014, the contents of all carotenoids are decreased proportionally to the decrease in chlorophyll content. It is shown that the rates of CO₂ assimilation and oxygen production by mutant chlorotica 2004 and 2014 plants are reduced. The quantum efficiency of photosynthesis in the mutants is 29–30% lower than in the control plants; in their hybrids, however, it is 1.5–2 times higher. It is proposed that both the greater role of dark respiration in gas exchange and the reduced photosynthetic activity in chlorotica mutants are responsible for the decreased phytomass increment in these plants. On the basis of these results, the conclusion is drawn that mutations chlorotica 2004 and 2014 affect the genes controlling the formation and functioning of various components of the photosynthetic apparatus.     

Purification of Protein Body-I of Rice Seed and its Polypeptide Composition

Protein body type one (PB-I) was isolated and purified fromdeveloping rice grain by a combination of sucrose density gradientcentrifugation and treatment with pepsin. SDS-PAGE analysisshowed that isolated PB-I contains several polypeptide groups,the largest having an apparent molecular size of 13 kDa andtwo smaller ones of 10 kDa and 16 kDa. The 13-kDa group wasfound to be composed of two polypeptides of slightly differentmolecular sizes, 13a (larger component) and 13b (smaller component).Most of the 13a and 13b polypeptides were shown to be largelyprolamins, although there were also some salt- and alcohol-insolublepolypeptides with an apparent molecular size of 13 kDa. It wasconcluded that PB-I is the accumulation site of rice prolamin.It was further estimated that the protein amount in PB-I accountedfor about 20% of the total protein of rice endosperm. (Received March 20, 1987; Accepted September 8, 1987)     

Polypeptide Composition of Photosynthetic Membranes from Chlamydomonas reinhardi and Anabaena variabilis

Anabaena variabilis, a blue-green alga lacking chlorophyll b, shows an absence of the major 22 and 24 kilodalton polypeptides which are present in the photosynthetic membranes of Chlamydomonas reinhardi and higher plants. These data are consistent with other investigations which have shown that these polypeptides are associated with chlorophyll b in the chloroplasts of higher plants, and indicate the presence of a light harvesting chlorophyll-protein complex in higher plants which contains the chlorophyll b of the photosynthetic membrane.     

Phytohormone Levels in Different Types of Symbiotic Mutants of Pea

The levels of the phytohormones auxin and gibberellin were studied in the original pea (Pisum sativumL.) cultivars Rondo and Ramonskii 77 and in different types of symbiotic mutants (non-nodulating, with single nodules, and supernodulating) induced from them. The results obtained indicated that the levels of the phytohormones in the symbiotic mutants depend on the plant's genotype, developmental phase, and infection with rhizobia. Two mutants were isolated whose phytohormonal status markedly differed from the original forms. These mutants may be used for identification of the genes that determine the auxin and gibberellin statuses.     

Insight into Poliovirus Genome Replication and Encapsidation Obtained from Studies of 3B-3C Cleavage Site Mutants

A poliovirus (PV) mutant (termed GG), which is incapable of producing 3AB, VPg, and 3CD proteins due to a defective cleavage site between the 3B and 3C proteins, replicated, producing 3BC-linked RNA rather than the VPg-linked RNA produced by the wild type (WT). GG PV RNA is quasi-infectious. The yield of infectious GG PV relative to replicated RNA is reduced by almost 5 logs relative to that of WT PV. Proteolytic activity required for polyprotein processing is normal for the GG mutant. 3BC-linked RNA can be encapsidated as efficiently as VPg-linked RNA. However, a step after genome replication but preceding virus assembly that is dependent on 3CD and/or 3AB proteins limits production of infectious GG PV. This step may involve release of replicated genomes from replication complexes. A pseudorevertant (termed EG) partially restored cleavage at the 3B-3C cleavage site. The reduced rate of formation of 3AB and 3CD caused corresponding reductions in the observed rate of genome replication and infectious virus production by EG PV without impacting the final yield of replicated RNA or infectious virus relative to that of WT PV. Using EG PV, we showed that genome replication and encapsidation were distinct steps in the multiplication cycle. Ectopic expression of 3CD protein reversed the genome replication phenotype without alleviating the infectious-virus production phenotype. This is the first report of a trans-complementable function for 3CD for any picornavirus. This observation supports an interaction between 3CD protein and viral and/or host factors that is critical for genome replication, perhaps formation of replication complexes.Poliovirus (PV) is the most extensively studied member of the picornavirus family and serves as a paradigm not only for picornaviruses but also for many of the nonretroviral positive strand RNA viruses (74). A schematic of the ∼7,500-nucleotide PV genome is shown in Fig. ​Fig.1A.1A. The 5′ end is linked covalently to a 22-amino-acid peptide termed VPg (virion protein genome linked) that is encoded by the 3B region of the genome. VPg and 3B are therefore used interchangeably. The 3′ end of the genome is terminated by a poly(rA) tail. Upon release of the genome into the host cell cytoplasm, genome translation is initiated by using the internal ribosome entry site. An ∼3,000-amino-acid polyprotein is produced. Complete cleavage of the polyprotein by virus-encoded proteases yields 10 proteins. The polyprotein can be divided further into three smaller polyproteins: P1, P2, and P3. P1 contains capsid proteins: VP0, VP3, and VP1. VP0 undergoes autocatalytic cleavage after genome encapsidation to produce VP4 and VP2 proteins. P2 performs host interaction functions required for robust virus multiplication, for example, shutoff of host cell translation and induction of vesicles employed for genome replication, the so-called replication complexes (RCs). P3 contains proteins that function most directly in genome replication, including the RNA-dependent RNA polymerase. Translation induces RCs, leading to genome replication. Early during infection, replicated genomes are employed as templates for translation, leading to an exponential amplification of RCs and replicated RNA. Ultimately, production of viral proteins ceases and replicated genomes are packaged. The use of RCs provides a barrier to genetic complementation; all proteins must be provided in cis, that is, produced from the RNA that they replicate.Open in a separate windowFIG. 1.PV genome organization and P3 processing pathway. (A) Schematic of the PV genome. The 5′ end of the genome is covalently linked to a peptide (VPg) encoded by the 3B region of the genome. The 3′ end contains a poly(rA) tail. Three cis-acting replication elements are known. oriL is located in 5′ NTR. oriR is located in the 3′ NTR. oriI is located in 2C-coding sequence for PV; the position of this element is virus dependent. oriI is the template for VPg uridylylation. Translation initiation employs an internal ribosome entry site (IRES). The single open reading frame encodes a polyprotein. P1 produces virion structural proteins as indicated. P2 produces proteins thought to participate in virus-host interactions required for genome replication. P3 produces proteins thought to participate directly in genome replication. Polyprotein processing is mediated by protease activity residing in 2A, 3C, and/or 3CD proteins. (B) Processing of the P3 precursor occurs by two independent pathways (60). There are major (I) and minor (II) pathways. In pathway I, processing between 3B and 3C yields 3AB and 3CD. In pathway II, processing between 3A and 3B yields 3A and 3BCD. 3BCD processing yields 3BC and 3D; 3BC processing yields 3B and 3C. Pathway II is proposed to function in genome replication and is not perturbed in the GG mutant.In addition to P3 proteins, genome replication requires three cis-acting replication elements (CREs): a cloverleaf structure located in the 5′ nontranslated region (NTR), termed oriL (left) (1, 5); a stem-loop structure located in 2C-coding sequence, termed oriI (internal) (30, 61); and a pseudoknot structure located in the 3′ NTR, termed oriR (right) (1, 40). The first step of genome replication is diuridylylation of VPg or a VPg-containing protein primer (62, 74). This reaction is templated by oriI but also requires oriL in a cell-free reaction and is catalyzed by the viral RNA-dependent RNA polymerase 3Dpol (4, 5, 11, 30, 61). In addition to the four terminal P3 cleavage products (3A, 3B, 3C, and 3D proteins) and the uncleaved P3 polyprotein, several “intermediates” are observed in infected cells (3AB, 3CD, and 3BCD proteins) (Fig. ​(Fig.1B)1B) (43, 57, 73). The major P3 cleavage pathway (I) produces 3AB and 3CD proteins; the minor P3 cleavage pathway (II) produces 3A and 3BCD proteins (Fig. ​(Fig.1B)1B) (60). In some cases, the intermediates have unique activities, specificities, and/or functions relative to their corresponding terminal cleavage products.Over the past 8 years much has been learned about oriI-templated VPg uridylylation in vitro for a variety of picornaviruses (28, 49, 53, 77, 92). However, it is still unclear whether or not VPg, 3BC(D), or 3AB is used in vivo to initiate genome replication. The VPg peptide can be uridylylated in vitro (62); however, VPg-pUpU does not chase efficiently into full-length RNA (81). 3BC(D) is uridylylated more efficiently than VPg in vitro, leading to the possibility that this precursor could be used in vivo (60). To date, 3AB has been uridylylated in vitro only in the presence of Mn²⁺ (66). In order to begin to probe the origin of VPg that is linked to picornaviral RNA, we created a PV mutant in which the cleavage site between 3B and 3C was changed from Gln-Gly to Gly-Gly (60). We refer to this mutant as GG. The GG mutation should be lethal for genome replication if use of the processed VPg peptide is absolutely required for genome replication. For the GG mutant, products of the major P3 cleavage pathway were no longer 3AB and 3CD but were now 3ABC and 3D instead. The kinetics of genome replication were reduced for the GG mutant relative to those for the wild type (WT). Surprisingly, the yield of replicated GG RNA was within an order of magnitude of that observed for WT RNA. Replicated GG RNA was then linked covalently to 3BC instead of VPg, as observed for WT PV. In spite of the substantial yield of replicated RNA, infectious virus was not recovered.We have performed a molecular characterization of the GG mutant. GG PV RNA is quasi-infectious. The low yield of virus recovery relative to replicated RNA reflects a block at a step in the PV multiplication cycle positioned after genome replication but prior to virus assembly. The existence of this step in the PV life cycle was suggested previously by Baltimore (8). Surprisingly, none of the defects associated with GG PV could be attributed to the absence of 3CD protease activity, suggesting that precursors larger than 3CD may be the primary proteases employed in vivo. All of the observed defects in GG PV multiplication were ameliorated in a pseudorevertant in which the 3B-3C cleavage site was changed from Gly-Gly to Glu-Gly. This mutant is referred to as EG. Molecular characterization of EG PV revealed for the first time a trans-complementable function for 3CD in genome replication. This observation supports a role for 3CD at a step preceding genome replication within RCs, perhaps RC formation. Our studies of EG PV confirmed the existence of a step between genome replication and virus assembly that requires 3CD and/or 3AB, thus providing compelling evidence for genome replication and genome encapsidation as distinct steps in the multiplication cycle. This study highlights the utility of polyprotein cleavage site mutants for evaluation of the viral multiplication cycle.     

Genetic Selection of Poliovirus 2Apro-Binding Peptides

The yeast two-hybrid system has been used to identify mammalian clones that interact with poliovirus 2A proteinase (2A^pro). Eight clones which encode previously unidentified human proteins were selected from a HeLa cell cDNA expression library. In addition, five clones encoding short peptides that interact with poliovirus 2A^pro were also identified. The lengths of these peptides range from 6 to 30 amino acids, but all of them contain the Leu-X-Thr-Z motif (X represents any amino acid; Z represents a hydrophobic residue). This sequence is invariably located just at the carboxy terminus of each peptide. This approach raises the possibility of designing substrate analogue inhibitors of 2A^pro. Thus, two nonhydrolyzable peptides containing the Leu-X-Thr-Z motif prevented cleavage of eukaryotic initiation factor 4G by poliovirus 2A^pro in vitro. A more general method for identifying peptides with antiproteinase activity is discussed.     

Inactivation and Heat Stabilization of Poliovirus by 2-Thiouracil

Treatment of poliovirus Type I with 10(-3)m 2-thiouracil (2-TU) resulted in the inactivation of greater than 90% of the virus infectivity and stabilization of approximately 50% of the residual virus to heat inactivation. These effects were due to a reaction with the protein moiety of the virus and could be blocked by pre-treatment of the virus with l-cystine or of the drug with cysteine. Both inactivation and stabilization occurred synchronously and reached equilibrium at the same time. Neither process was reversed by reducing agents. It is suggested that an oxidized form of 2-TU reacts with capsid sulfhydryl groups to form a product which is stable in either the inactive or heat-resistant form.     

Genetic Analysis of Petite Mutants of SACCHAROMYCES CEREVISIAE : Transmissional Types

We have studied a number of petite [rho^- ] mutants of Saccharomyces cerevisiae induced in a wild-type strain of mitochondrial genotype [ome^- CHL ^R ERY^S OLI^S_1,2,3 PAR^S] by Berenil and ethidium bromide, all of which have retained two mitochondrial genetic markers, [CHL^R] and [ERY^S], but have lost all other known markers. Though stable in their ability to retain these markers in their genome, these mutants vary widely among themselves in suppressiveness and in the extent to which the markers are transmitted on crossing to a common wild-type tested strain. In appropriate crosses all of the strains examined in this study demonstrate mitochondrial polarity, and thus have also retained the [ome^-] locus in a functional form; however, five different transmissional types were obtained, several of them quite unusual, particularly among the strains originally induced by Berenil. One of the most interesting types is the one that appears to reverse the parental genotypes with [CHL^R ERY^S] predominating over [CHL^S ERY^R] in the diploid [rho+] progeny, rather than the reverse, which is characteristic of analogous crosses with [rho+] or other petites. Mutants in this class also exhibited low or no suppressiveness. Since all of the petites reported here are derived from the same wild-type parent, and so have the same nuclear background, we have interpreted the transmissional differences as being due to different intramolecular arrangements of largely common retained sequences.