DNA sequences of polyoma virus early deletion mutants.

The DNA sequences of four "early" viable deletion mutants of polyoma virus have been determined. Two of these (dl-8 and dl-23) are mutants with deletions in the region of the genome that codes for parts of both large and middle T-antigens, and two (dl-6 and dl-28) are mutants with deletions around the viral origin of replication. The former mutants have altered transformation properties relative to wild-type virus, and dl-8 appears to be replication deficient (B. E. Griffin and C. Maddock, J. Virol. 31:645-656, 1979). Sequences are discussed in terms of the altered phenotypes observed for the various mutants, the DNA structures and protein sequences that are affected by the deletions, and how these might affect the biological properties of the mutants.     

Viral DNA Synthesis in Cells Infected by Temperature-Sensitive Mutants of Simian Virus 40

Temperature-sensitive mutants of simian virus 40 (SV40) have been classified as those that are blocked prior to viral DNA synthesis at the restrictive temperature, "early" mutants, and those harboring a defect later in the replication cycle, "late" mutants. Mutants of the A and D complementation groups are early, those of the B, C, and BC groups are late. Our results confirm earlier reports that A mutants are defective in a function required for the initiation of each round of viral DNA synthesis. D mutants, on the other hand, continue viral DNA replication at the restrictive temperature after preincubation at the permissive temperature. The length of time required for D function to be expressed at the permissive temperature-after which infection proceeds unabated on shifting of the cultures to the restrictive temperature-is 10 to 20 h. The viral DNA synthesized in D mutants under these conditions progresses in normal fashion through replicative intermediate molecules to mature component I and II DNA molecules.     

Activation of protein kinase Tel1 through recognition of protein-bound DNA ends

Double-strand breaks (DSBs) in chromosomal DNA elicit a rapid signaling response through the ATM protein kinase. ATM corresponds to Tel1 in budding yeast. Here we show that the catalytic activity of Tel1 is altered by protein binding at DNA ends via the Mre11-Rad50-Xrs2 (MRX) complex. Like ATM, Tel1 is activated through interaction with the MRX complex and DNA ends. In vivo, Tel1 activation is enhanced in sae2Δ or mre11-3 mutants after camptothecin treatment; both of these mutants are defective in the removal of topoisomerase I from DNA. In contrast, an sae2Δ mutation does not stimulate Tel1 activation after expression of the EcoRI endonuclease, which generates "clean" DNA ends. In an in vitro system, tethering of Fab fragments to DNA ends inhibits MRX-mediated DNA end processing but enhances Tel1 activation. The mre11-3 mutation abolishes DNA end-processing activity but does not affect the ability to enhance Tel1 activation. These results support a model in which MRX controls Tel1 activation by recognizing protein-bound DNA ends.     

Two naturally occurring deletion mutants of 12S seed storage proteins in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Two naturally occurring Arabidopsis mutants, Cape Verde Islands and Monte (Mr-0), with aberrant 12S seed storage protein (SSP) profiles have been identified by SDS-PAGE. In both mutants, one of the 12S globulin bands is missing while a new band of lower molecular mass is present. Tandem mass spectrometry-mass spectrometry (MS/MS) analyses of the mutant peptides have revealed that both are shorter variants of 12S globulin with deletion sites detected within the -subunits of 12S globulin cruciferin B (CRB) and C (CRC), respectively. Sequence analyses of the genomic DNA flanking the deletion sites have demonstrated that both deletions occurred at the genomic level. These two mutants are referred to as CRB12 and CRC13 with the delta sign indicating a deletion and the number indicating amino acids deleted. Alignment of these two mutant sequences with that of soybean A3B4 subunit, for which the crystal structure was determined recently, have revealed that the CRC13 deletion is located in a hypervariable/disordered region, and will probably not affect the structure of the hexameric globulin. The CRB12 deletion, however, is located in a binding region that is thought to be important for the hexamer formation. However, CRB12 appears to accumulate normally as judged by its band intensity relative to the other SSP subunits on the protein gels. Thus it seems that the seed can, to a certain extent, tolerate some mutations in its storage proteins.     

黄瓜花叶病毒卫星RNA人工突变体的构建及其侵染性测定

从1个369nt的黄瓜花叶病毒(Cucumbermosaicvirus,CMV)卫星RNA的cDNA出发,采用DNA改组技术构建人工突变体,经过体外转录,将其与不携带卫星RNA的黄瓜花叶病毒株进行假重组,鉴定突变体的生物活性,结果显示所获得的4个卫星RNA的点突变子MS1、MS5、MS6和MS11中,只有MS11仍然具有复制能力;而其他3个点突变子,尽管均只有1个位点的替换,却不能在辅助病毒作用下复制。序列比较分析发现MS11的突变位点位于卫星RNA变异区内,发生的突变与自发突变一致,其他3个突变子的突变位点发生在卫星RNA的高度保守区。而且通过侵染性试验证实突变子MS11与野生型Yi没有明显的差异。由此可推测卫星RNA序列中的高度保守区与卫星RNA的生物活性密切相关,个别碱基的突变会导致RNA二级结构的改变,进而引起其复制能力或稳定性的完全丧失。     

A comparison of direct infusion MS and GC-MS for metabolic footprinting of yeast mutants

Recent technical advances in mass spectrometry (MS) have propelled this technology to the forefront of methods employed in metabolome analysis. Here, we compare two distinct analytical approaches based on MS for their potential in revealing specific metabolic footprints of yeast single-deletion mutants. Filtered fermentation broth samples were analyzed by GC-MS and direct infusion ESI-MS. The potential of both methods in producing specific and, therefore, discriminant metabolite profiles was evaluated using samples from several yeast deletion mutants grown in batch-culture conditions with glucose as the carbon source. The mutants evaluated were cat8, gln3, ino2, opi1, and nil1, all with deletion of genes involved in nutrient sensing and regulation. From the analysis, we found that both methods can be used to classify mutants, but the classification depends on which metabolites are measured. Thus, the GC-MS method is good for classification of mutants with altered nitrogen regulation as it primarily measures amino acids, whereas this method cannot classify mutants involved in regulation of phospholipids metabolism as well as the direct infusion MS (DI-MS) method. From the analysis, we find that it is possible to discriminate the mutants in both the exponential and stationary growth phase, but the data from the exponential growth phase provide more physiological relevant information. Based on the data, we identified metabolites that are primarily involved in discrimination of the different mutants, and hereby providing a link between high-throughput metabolome analysis, strain classification, and physiology.     

DNA infectivity and the induction of host DNA synthesis with temperature-sensitive mutants of simian virus 40.

Host DNA synthesis is induced when CV-1 (monkey kidney) cell cultures are infected at 40 C with wild-type virions or with temperature-sensitive Simian virus 40 mutants of the "early" complementation group A. Host DNA synthesis is not induced when cultures are infected with mutants of the late complementation group D. The simplest explanation for these observations, that induction depends not upon the expression of some early gene function but rather on the presence of an active D protein in the infecting virion, has been examined. Indirect experiments suggest that this explanation is not correct. Moreover, the induction of host DNA synthesis is impaired when cultures are infected with mutants of the A group at 42.5 C rather than 40 C, suggesting that the A function may be responsible for host induction. The inability of D virions to induce host DNA synthesis may reflect their inability to "uncoat" at 40C.     

Herpes simplex virus type 1 alkaline nuclease is required for efficient processing of viral DNA replication intermediates.

Mutations in the alkaline nuclease gene of herpes simplex type 1 (HSV-1) (nuc mutations) induce almost wild-type levels of viral DNA; however, mutant viral yields are 0.1 to 1% of wild-type yields (L. Shao, L. Rapp, and S. Weller, Virology 195:146-162, 1993; R. Martinez, L. Shao, J.C. Bronstein, P.C. Weber, and S. Weller, Virology 215:152-164, 1996). nuc mutants are defective in one or more stages of genome maturation and appear to package DNA into aberrant or defective capsids which fail to egress from the nucleus of infected cells. In this study, we used pulsed-field gel electrophoresis to test the hypothesis that the defects in nuc mutants are due to the failure of the newly replicated viral DNA to be processed properly during DNA replication and/or recombination. Replicative intermediates of HSV-1 DNA from both wild-type- and mutant-infected cells remain in the wells of pulsed-field gels, while free linear monomers are readily resolved. Digestion of this well DNA with restriction enzymes that cleave once in the viral genome releases discrete monomer DNA from wild-type virus-infected cells but not from nuc mutant-infected cells. We conclude that both wild-type and mutant DNAs exist in a complex, nonlinear form (possibly branched) during replication. The fact that discrete monomer-length DNA cannot be released from nuc DNA by a single-cutting enzyme suggests that this DNA is more branched than DNA which accumulates in cells infected with wild-type virus. The well DNA from cells infected with wild-type and nuc mutants contains XbaI fragments which result from genomic inversions, indicating that alkaline nuclease is not required for mediating recombination events within HSV DNA. Furthermore, nuc mutants are able to carry out DNA replication-mediated homologous recombination events between inverted repeats on plasmids as evaluated by using a quantitative transient recombination assay. Well DNA from both wild-type- and mutant-infected cells contains free U(L) termini but not free U(S) termini. Various models to explain the structure of replicating DNA are considered.     

Single particle analysis of thylakoid proteins from Thermosynechococcus elongatus and Synechocystis 6803: localization of the CupA subunit of NDH-1

The larger protein complexes of the cyanobacterial photosynthetic membrane of Thermosynechoccus elongatus and Synechocystis 6803 were studied by single particle electron microscopy after detergent solubilization, without any purification steps. Besides the "standard" L-shaped NDH-1L complex, related to complex I, large numbers of a U-shaped NDH-1MS complex were found in both cyanobacteria. In membranes from Synechocystis DeltacupA and DeltacupA/cupB mutants the U-shaped complexes were absent, indicating that CupA is responsible for the U-shape by binding at the tip of the membrane-bound arm of NDH-1MS. Comparison of membranes grown under air levels of CO(2) or 3% CO(2) indicates that the number of NDH-1MS particles is 30-fold higher under low-CO(2).     

Molecular mechanism and energetics of clamp assembly in Escherichia coli. The role of ATP hydrolysis when gamma complex loads beta on DNA

Escherichia coli DNA polymerase III holoenzyme is a multisubunit composite containing the beta sliding clamp and clamp loading gamma complex. The gamma complex requires ATP to load beta onto DNA. A two-color fluorescence spectroscopic approach was utilized to study this system, wherein both assembly (red fluorescence; X-rhodamine labeled DNA anisotropy assay) and ATP hydrolysis (green fluorescence; phosphate binding protein assay) were simultaneously measured with millisecond timing resolution. The two temporally correlated stopped-flow signals revealed that a preassembled beta. gamma complex composite rapidly binds primer/template DNA in an ATP hydrolysis independent step. Once bound, two molecules of ATP are rapidly hydrolyzed (approximately 34 s(-1)). Following hydrolysis, gamma complex dissociates from the DNA ( approximately 22 s(-1)). Once dissociated, the next cycle of loading is severely compromised, resulting in steady-state ATP hydrolysis rates with a maximum of only approximately 3 s(-1). Two single-site beta dimer interface mutants were examined which had impaired steady-state rates of ATP hydrolysis. The pre-steady-state correlated kinetics of these mutants revealed a pattern essentially identical to wild type. The anisotropy data showed that these mutants decrease the steady-state rates of ATP hydrolysis by causing a buildup of "stuck" binary-ternary complexes on the primer/template DNA.     

Partially deficient methylation of cytosine in DNA at CCATGG sites stimulates genetic recombination of bacteriophage lambda

Lambda bacteriophages grown on arl mutants of Escherichia coli ("Arl-" phages) display intermediate levels of cytosine methylation: less 5-methylcytosine (m5C) than phages grown on wild-type bacteria ("Arl+" phages) but more than phages grown on dcm mutants, and thus lacking the methylated sequences (Cm5CATGG) characteristic of E. coli K-12 bacteria ("Dcm-" phages). "Arl-" phages are one twelfth as resistant to Eco RII restriction (recognition site CCATGG) as "Arl+" phages, but 40-fold more resistant than "Dcm-" phages. Chromatographic analyses show the 5-methylcytosine content of "Arl-" DNA to be one third that of "Arl+" DNA. Altered cytosine methylation frequency correlates with two previously described properties of "Arl-" phages, increased genetic recombination and unusual sensitivity of phage DNA to endonuclease S1, which are absent in phages grown on dcm or dcm arl bacteria. Methylated/unmethylated heteroduplex DNA prepared in vitro (one strand from Eco RII-modified phages/one from "Dcm-" phages) is highly recombinogenic but not S1-sensitive. We hypothesize that hemimethylated CCATGG sites in "Arl-" DNA are necessary and sufficient for enhanced recombination, and necessary but not sufficient for S1 sensitivity.     

Expression of catalytic mutants of the mtDNA helicase Twinkle and polymerase POLG causes distinct replication stalling phenotypes

The mechanism of mitochondrial DNA replication is a subject of intense debate. One model proposes a strand-asynchronous replication in which both strands of the circular genome are replicated semi-independently while the other model proposes both a bidirectional coupled leading- and lagging-strand synthesis mode and a unidirectional mode in which the lagging-strand is initially laid-down as RNA by an unknown mechanism (RITOLS mode). Both the strand-asynchronous and RITOLS model have in common a delayed synthesis of the DNA-lagging strand. Mitochondrial DNA is replicated by a limited set of proteins including DNA polymerase gamma (POLG) and the helicase Twinkle. Here, we report the effects of expression of various catalytically deficient mutants of POLG1 and Twinkle in human cell culture. Both groups of mutants reduced mitochondrial DNA copy number by severe replication stalling. However, the analysis showed that while induction of POLG1 mutants still displayed delayed lagging-strand synthesis, Twinkle-induced stalling resulted in maturated, essentially fully double-stranded DNA intermediates. In the latter case, limited inhibition of POLG with dideoxycytidine restored the delay between leading- and lagging-strand synthesis. The observed cause-effect relationship suggests that Twinkle-induced stalling increases lagging-strand initiation events and/or maturation mimicking conventional strand-coupled replication.     

Assembly of the mitochondrial membrane system: isolation of mitochondrial transfer ribonucleic acid mutants and characterization of transfer ribonucleic acid genes of Saccharomyces cerevisiae

A method is described for isolating cytoplasmic mutants of Saccharomyces cerevisiae with lesions in mitochondrial transfer ribonucleic acids (tRNA's). The mutants were selected for slow growth on glycerol and for restoration of wild-type growth by cytoplasmic "petite" testers that contain regions of mitochondrial deoxyribonucleic acid (DNA) with tRNA genes. The aminoacylated mitochondrial tRNA's of several presumptive tRNA mutants were analyzed by reverse-phase chromatography on RPC-5. Two mutant strains, G76-26 and G76-35, were determined to carry mutations in the cysteine and histidine tRNA genes, respectively. The cysteine tRNA mutant was used to isolate cytoplasmic petite mutants whose retained segments of mitochondrial DNA contain the cysteine tRNA gene. The segment of one such mutant (DS504) was sequenced and shown to have the cysteine, histidine, and threonine tRNA genes. The structures of the three mitochondrial tRNA's were deduced from the DNA sequence.     

A new RNA vaccine platform based on MS2 virus-like particles produced in Saccharomyces cerevisiae

mRNA vaccines are potentially attractive alternatives to DNA vaccines more often discussed, as they are generally considered safer than their DNA counterparts. The major limitations on the potency of RNA vaccines are their instability and inability to spread in vivo. Virus-like particles (VLPs) based on the bacteriophage MS2 have demonstrated remarkably high stability and may provide an improved platform for RNA-based genetic vaccination. However, no in vivo study of an MS2 VLP-mediated RNA vaccine has been reported. Therefore, we developed a model vaccine wherein MS2 VLPs packaging HIV-1 gag mRNAs (1544 bases) were produced in Saccharomyces cerevisiae, and then, used to immunize BALB/c mice. Serological analyses showed that antigen-specific antibody responses were elicited by immunization. These findings suggest that MS2 VLPs can be used in the design and construction of novel and safe phage-based mRNA delivery vectors.     

Lethality of rep recB and rep recC double mutants of Escherichia coli

A rep mutation in combination with a recB or a recC mutation renders Escherichia coli non-viable. This conclusion is based on the following lines of evidence: (i) double mutants cannot be constructed by P1 transduction; (ii) induction of the λ Gam protein, which inactivates most of the RecBCD activities, is lethal in rep mutants; (iii) rep recBts recCts mutants are not viable at high temperature. The reasons for a requirement for the RecBCD enzyme in rep strains were investigated. Initiation of chromosome replication, elongation and chromosomal segregation do not seem impaired in the rep recBts recCts mutant at the non-permissive temperature. The viability of other rep derivatives was tested. rep recA recD triple mutants are not viable, whereas rep recD and rep recA double mutants are. Inactivation of both exoV activity and recBC -dependent homologous recombination is therefore responsible for the non-viability of rep recBC strains. However, sbcA and sbcB mutations, which render recBC mutants recombination proficient, do not restore viability of rep recBC mutants, indicating that recombination via the RecF or the RecE pathways cannot functionally replace RecBCD-mediated recombination. The specific requirement for RecBCD suggests the occurrence of double-strand DNA breaks in rep strains. Additional arguments in favour of the presence of DNA lesions in rep mutants are as follows: (i) expression of SOS repair functions delays lethality of rep derivatives after inactivation of RecBCD; (ii) sensitivity of rep strains to ultraviolet light is increased by partial inactivation of RecBCD. A model for the recovery of cells from double-strand breaks in rep mutants is discussed.     

The product of the pilQ gene is essential for the biogenesis of type IV pili in Neisseria gonorrhoeae

The product of the Neisseria gonorrhoeae omc gene possesses regions homologous to those found in members of a protein superfamily that are associated with the translocation of proteins and DNA-protein complexes across the outer membrane. Amongst its protein homologues, Omc has higher overall homology to PilQ, which is required for type IV pilus expression in Pseudomonas aeruginosa , and OrfE, which is required for sequence-specific DNA uptake by Haemophilus influenzae . The function of Omc, however, is unknown and gonococcal omc mutants have not been described. We constructed gonococcal mutants expressing truncated forms of the protein, and found that these mutants are severely defective for both pilus expression and competence for natural transformation. To be consistent with pre-existing pilus gene nomenclature, we have redesignated the gene pilQ instead of omc , and its product, PilQ instead of Omc. The MS11 gene was sequenced and found to differ from the DNA sequence reported for that of another gonococcal strain; these differences were associated with a repeated DNA element, suggesting a genetic basis for structural variation in PilQ. The results also show that PilQ⁻ mutants are distinct from previously described gonococcal pilus-assembly mutants and P. aeruginosa PilQ⁻ mutants by virtue of their expression of rare pilus filaments. Taking these data into account, PilQ is proposed to function in the terminal steps of organelle biogenesis by acting as a pilus channel or pore.     

Isolation of four phenotypic classes of transfer-proficient, pilus-specific-bacteriophage-resistant mutants of an F-like plasmid of Escherichia coli K12

Summary 173 independent mutants of phenotype Tra⁺ MS2^R were isolated from the derepressed F-like R-plasmid, R192-7, of Escherichia coli K12. Various tests of transfer frequency and phage infectivity of 21 of these mutants grouped them into four clearly distinct phenotypic classes. Class (1): mutants of high transfer frequency proficient in MS2 adsorption but to various extents defective in phage penetration, showing resistance or partial sensitivity to both MS2 and M13 equally. Clss (2): mutants of high transfer frequency which fail to adsorb MS2, resistant to both MS2 and M13. Class (3): mutants of reduced transfer frequency, approximately 10^-2 of that of R192-7, resistant to both MS2 and M13. Class (4): mutants of low transfer frequency, approximately 10^-3 to 10^-4 of that of R192-7 and resembling that of the repressed wild type R192, resistant to both MS2 and M13. Pili of mutants of classes (1) and (2) showed structural abnoramlities in electron microscopy.