The S and U genes of bacteriophage mu are located in the invertible G segment of mu DNA.

An F′ plasmid containing only the β and G segments of bacteriophage Mu DNA in a β-G-β structure was isolated as a LacZ^? segregant of an F′lac plasmid containing Mu prophages inserted in the lacI and lacY genes. The segregant arose by homologous recombination between the similarly oriented G regions located in oppositely oriented Mu prophages whose α segments were directed toward the lacZ gene. Electron microscopic observation of single-stranded plasmid DNA from the segregant revealed the expected β-G-β stem and loop structure in which the double-stranded stem was the same length as, β and the single-stranded loop was the same length as G. Marker rescue experiments with Mu amber mutants defective in each of the known essential Mu genes showed that strains carrying this F′-β-G-β plasmid contained the wild-type alleles for S and U mutations but not for mutations in other genes. more detailed mapping of the R S U region by deletion mapping in Mu prophages and λpMu transducing phages produced two gene orders, R S U and R U S, thus indicating that the S and U genes were located in the invertible G segment rather than in β. Confirmation of this conclusion was obtained from results of DNA heteroduplexing experiments which showed that deletions ending within gene U end physically within G segment DNA. The location of essential genes S and U within G and the location and extent of G segment DNA which is nonessential for growth (L. T. Chow, R. Kahmann, and D. Kamp, 1977, J. Mol. Biol., 113, 591–609), taken together, reveal that the order of genes in viable Mu phage with G in the G(+) orientation is R S U.     

Analysis of interactions among factors involved in the bacteriophage T3 DNA packaging reaction in a defined in vitro system

During head assembly of phage T3, DNA is packaged into the cavity of a preformed protein shell, called the prohead, with the aid of noncapsid, packaging proteins, the products of genes 18 and 19 (gp18 and gp19). gp18 and gp19 separately form complexes with DNA and proheads, respectively. These complexes associate to form a precursor which can be converted to filled heads by the addition of ATP. Interactions among factors involved in DNA packaging were analyzed. In the presence of ATP, gp19 formed functional complexes with proheads. Formation of gp19-prohead complex showed a sigmoidal dependence on ATP concentration with a half maximal concentration of about 7.5 microM. Six molecules of gp19 bound to the prohead at a saturating amount of gp19. gp19 did not bind to proheads lacking the connector of gp8 (8- prohead). In the absence of ATP, proheads were inactivated by gp19. The gp19-prohead complexes formed in the absence of ATP contained 20-30 gp19 molecules per prohead and formed multimeric aggregates. 8- proheads did not bind gp19 and did not form such aggregates even in the absence of ATP. From these results, we conclude that 6 molecules of gp19 bind to the gp8 connector structure in the portal vertex of the prohead. The cleavage patterns of gp19 by several proteases were altered by the addition of ATP, indicating that ATP induces a conformational change in gp19, gp18 bound only to linear, duplex DNA.     

Cloning and sequencing of the genetic right end of bacteriophage T3 DNA

The genetic right end of phage T3 DNA, from the beginning of gene 17, was cloned and sequenced. Genes 17, 18, and 19 were identified by comparing the sequence with the genetic map and by comparing the calculated and observed molecular weights of gene products. N-terminal amino acid sequence of the gene 17 product (gp17) predicted from the nucleotide sequence was consistent with the data from the analysis of purified gp17. Gene 17.5 was identified as the lysis gene on the basis of the presence of a nonsense codon within an open reading frame in the sequence of DNA from an amber mutant of lysis gene. In addition, five potential genes have been identified. Sequences corresponding to a promoter for phage T7 RNA polymerase (Rosa and Andrews, 1981) and to a class-III promoter for phage T3 RNA polymerase (Sarker et al., 1985) were found. The genomic organization and the nucleotide and deduced amino acid sequences of T3 were compared with those of T7. The genomic organizations of T3 and T7 were identical in this region. The sequence comparisons of T3 and T7 DNA point out the highly conserved sequences in all genes but also heavily varied regions in some genes. From these comparisons, possible implications with regard to structural and functional domains within several genes are discussed.     

7型腺病毒疫苗株DNA左侧17.5 mu片段克隆及4.8 mu片段核苷酸序列分析

目的获得７型腺病毒疫苗株ＤＮＡ左侧０～１７５ｍｕ片段克隆,分析０～４８ｍｕ片段（末端倒置重复序列、包装信号位点和Ｅｌａ区）核苷酸序列。方法从Ａｄ７疫苗株感染的Ａ５４９细胞提取Ａｄ７ＤＮＡ,将其０３～１７５ｍｕ片段克隆到质粒ｐＡｄ７Ｔ,并用自动和银染方法测序。结果获得了Ａｄ７疫苗株左侧１７５ｍｕ片段克隆,测定了左侧１７３７ｂｐ核苷酸序列,其中Ｅｌａ区所编码的６３００、２４０００、２８０００等３个蛋白的ＤＮＡ序列,与Ｇｏｍｅｎ株的同源性分别为９８９％、９７３％和９７５％,推导编码氨基酸的同源性分别为９６６％、９６５％和９６９％。这３个蛋白与Ｇｒｉｄｅｒ株的同源性分别为１００％、９９７％、和９９７％;推导编码氨基酸的同源性分别为１００％、９９１％和９９２％。结论Ａｄ７疫苗株左侧１７３８ｂｐ核苷酸与Ａｄ７Ｇｏｍｅｎ株和Ｇｒｉｄｅｒ株相应片段,具有高度的同源性。     

外显子组测序是发现单基因病致病基因的捷径

新一代测序技术的产生和发展为疾病研究带来了新的机遇.作为一种高效、快速和高性价比的研究方法,外显子组测序技术已经开始应用于单基因疾病等遗传病的研究.该技术已经得到国际学术界的广泛认可,也将越来越多地应用于单基因疾病的研究中.这将对单基因病致病基因的发现产生巨大推动作用.作者对国内外近两年应用外显子测序技术检测疾病相关基因的一些研究进行文献综述.     

On the molecular mechanism of DNA translocation during in vitro packaging of bacteriophage T3 DNA

The process of packaging of bacteriophage T3 DNA in a defined in vitro system can be separated into two stages: formation of a precursor complex (50 S complex) in the presence of adenosine-5'-O-(3'-thiotriphosphate) (ATP-gamma-S) and subsequent translocation of DNA into the head by the addition of ATP. Packaged DNA exits when DNA translocation is interrupted by the addition of ATP-gamma-S (M. Shibata, H. Fujisawa, and T. Minagawa, 1987, Virology, in press; M. Shibata, H. Fujisawa, and T. Minagawa, 1987, J. Mol. Biol., in press). The in vitro system packaged nicked and cross-linked DNAs but did not package single-stranded DNA. DNA packaging was inhibited by intercalating reagents such as ethidium bromide, acridine orange, and 4',6-diamino-2-phenylindole dihydrochloride. The inhibitory effect was proportional to the ability of intercalating agents to unwind DNA. Ethidium bromide did not inhibit the formation of 50 S complex but blocked translocation of DNA into and out of the capsid. DNA packaging was inhibited by actinomycin D and distamycin A which bind to the minor groove of the DNA helix. From these results, we conclude that DNA packaging mechanism utilizes the exterior structure of duplex DNA for translocating the DNA into the capsid.     

Circular monomers of bacteriophage lambda DNA as substrates for in vitro packaging

Monomeric circular λ DNA molecules were isolated from phage-infected cells, purified, and their biological activity tested using in vitro DNA packaging and λ terminase assays. These circles, which contain a single cos, proved to be as efficient as linear λ concatemers both for packaging into viable phage and cleavage at cos by highly purified λ terminase.     

Comprehensive massive parallel DNA sequencing strategy for the genetic diagnosis of the neuro-cardio-facio-cutaneous syndromes

Variants in 11 genes of the RAS/MAPK signaling pathway have been causally linked to the neuro-cardio-facio-cutaneous syndromes group (NCFCS). Recently, A2ML1 and RIT1 were also associated with these syndromes. Because of the genetic and clinical heterogeneity of NCFCS, it is challenging to define strategies for their molecular diagnosis. The aim of this study was to develop and validate a massive parallel sequencing (MPS)-based strategy for the molecular diagnosis of NCFCS. A multiplex PCR-based strategy for the enrichment of the 13 genes and a variant prioritization pipeline was established. Two sets of genomic DNA samples were studied using the Ion PGM System: (1) training set (n=15) to optimize the strategy and (2) validation set (n=20) to validate and evaluate the power of the new methodology. Sanger sequencing was performed to confirm all variants and low covered regions. All variants identified by Sanger sequencing were detected with our MPS approach. The methodology resulted in an experimental approach with a specificity of 99.0% and a maximum analytical sensitivity of ≥98.2% with a confidence of 99%. Importantly, two patients (out of 20) harbored described disease-causing variants in genes that are not routinely tested (RIT1 and SHOC2). The addition of less frequently altered genes increased in ≈10% the diagnostic yield of the strategy currently used. The presented workflow provides a comprehensive genetic screening strategy for patients with NCFCS in a fast and cost-efficient manner. This approach demonstrates the potential of a combined MPS–Sanger sequencing-based strategy as an effective diagnostic tool for heterogeneous diseases.     

A defined in vitro system for packaging of bacteriophage T3 DNA

Using purified components, we have constructed an in vitro system for packaging of mature phage T3 DNA. In addition to mature T3 DNA, the system contained T3 proheads and the products of gene 18 (gp18) and gene 19 (gp19). The reaction required Mg2+, ATP, and polyvinyl alcohol. Spermidine was stimulatory but not absolutely required for the packaging reaction. Polyvinyl alcohol could be replaced by polyethylene glycol. The packaging efficiency decreased with decreasing molecular weight of the polymer, and low molecular weight polyols such as sucrose, sorbitol, and glycerol were inactive. The packaging reaction exhibited a sigmoidal relationship with respect to the concentration of ATP with the concentration for half maximal activity about 15 microM. A nonhydrolyzable ATP analog, adenosine 5'-O-(3-thiotriphosphate), inhibited the packaging reaction.     

7型腺病毒疫苗株DNA左侧17.5mu片段克隆及4.8mu片？…

目的 获得７型腺病毒疫苗株ＤＮＡ左侧０－１７．５ｍｕ片段克隆,分析０－４．８ｍｕ片段（末端倒置重复序列,包装信号位点和Ｅｌａ区）核苷酸序列,。方法从Ａｄ７疫苗株感染的Ａ５４９细胞提取Ａｄ７ ＤＮＡ,将其０．３－１７．５ｍｕ片段克隆到质粒ｐＡｄ７Ｔ,并用自动和银染方法测序。结果 获得了Ａｄ７疫苗株左侧１７．５ｍｕ片段克隆,测定了左侧１７３７ｂｐ核苷酸序列,其中Ｅｌａ区所编码的６３００,２４０００,２     

In vitro binding of anthrax protective antigen on bacteriophage T4 capsid surface through Hoc-capsid interactions: a strategy for efficient display of large full-length proteins

An in vitro binding system is described to display large full-length proteins on bacteriophage T4 capsid surface at high density. The phage T4 icosahedral capsid features 155 copies of a nonessential highly antigenic outer capsid protein, Hoc, at the center of each major capsid protein hexon. Gene fusions were engineered to express the 83-kDa protective antigen (PA) from Bacillus anthracis fused to the N-terminus of Hoc and the 130-kDa PA-Hoc protein was expressed in Escherichia coli and purified. The purified PA-Hoc was assembled in vitro on hoc(-) phage particles. Binding was specific, stable, and of high affinity. This defined in vitro system allowed manipulation of the copy number of displayed PA and imposed no significant limitation on the size of the displayed antigen. In contrast to in vivo display systems, the in vitro approach allows all the capsid binding sites to be occupied by the 130-kDa PA-Hoc fusion protein. The PA-T4 particles were immunogenic in mice in the absence of an adjuvant, eliciting strong PA-specific antibodies and anthrax lethal toxin neutralizing antibodies. The in vitro display on phage T4 offers a novel platform for potential construction of customized vaccines against anthrax and other infectious diseases.     

The orderly, in vitro emergence of DNA from bacteriophage phi29 particles

phi29 DNA-containing 12-13- particles (produced by infecting nonsuppressor hosts of Bacillus subtilis with phage containing suppressible mutations in cistrons 12 and 13) can be complemented with lysates containing proteins p12* and p13 to yield infectious phage. Complementation of these particles with lysates containing p12* but not p13 or complementation with purified p12* in the absence of p13 produces a structure (called complex) which has a markedly different organization. Electron microscopy and sedimentation analysis after digestion with DNase I or proteinase K indicate that complex is composed of an intact phage head with a genome-sized linear DNA molecule attached at the collar-tail region. EcoRI digestion establishes that the DNA molecule has a unique orientation. Gel electrophoresis indicates that p12*, the neck appendage protein, is transferred to the particles when complex is formed. Complex can also be produced by incubation of 12-13- particles at 42 degrees , by incubation at pH 6.0, or by incubation in the presence of 20 mM EDTA. Complex is also formed from DNA-containing 12- particles but to a lesser extent.     

Characterization of preearly genes in the terminal repetition of bacteriophage BF23 DNA by nucleotide sequencing and restriction mapping

A finer restriction map of the terminal repetition of bacteriophage BF23 DNA was determined and used to localize a 3.4-kbp deletion in the terminal repetition and to determine the physical location of preearly gene A2-A3. The nucleotide sequence of gene A2-A3 was determined and shown to code for a protein of 125 amino acids with no indication of a membrane transport sequence. The beginning of an adjacent gene, probably gene A1, was also sequenced.     

Early events in DNA packaging in a defined in vitro system of bacteriophage T3

We have developed a defined in vitro system for packaging phage T3 DNA which is composed of purified proheads and the noncapsid proteins gp18 and gp19, products of genes 18 and 19. The reaction requires Mg2+, ATP, and polyethylene glycol and is inhibited by a nonhydrolyzable ATP analog, adenosine-5'-O-(3'-thiotriphosphate) (ATP-gamma-S) (K. Hamada, H. Fujisawa, and T. Minagawa, 1986, Virology 151, 119-123). About 30% of added mature T3 DNA was packaged into heads in the defined system. A complex with a sedimentation coefficient of about 50 S (50 S complex) accumulated in the reaction mixture containing ATP-gamma-S. The 50 S complex was DNase sensitive and was converted to filled heads by a second reaction in the presence of ATP without addition of DNA, proheads, gp18, and gp19. These results indicate that during early stages of DNA packaging, formation of precursor complexes proceeds by an allosteric mechanism with ATP acting as effector. The movement of DNA into the head is driven by the energy released by hydrolysis of ATP. gp18 formed a complex with DNA without addition of ATP-gamma-S and gp19. gp18-DNA complex was DNase sensitive and did not bind gp19; it was converted to filled heads by way of a second reaction after addition of ATP, gp19, and proheads. gp19 formed a functional complex with prohead in the presence of ATP-gamma-S or ATP. The complex did not bind gp18 but was converted to filled heads by incubation with ATP, gp18, and DNA. In the absence of ATP-gamma-S, gp19 formed complexes with prohead that were abortive in DNA packaging. Formation of the 50 S complex occurred in a reaction mixture containing gp18-DNA and gp19-prohead complexes in the presence of ATP-gamma-S. From these results, we propose details of the molecular mechanism of DNA packaging in the defined in vitro system.     

In vitro insertions and deletions in the G segment of phage mu DNA do not abolish the inversion process

The growth of rabies virus at a low pH of the culture medium resulted in the production of a large number of spikeless virions which lacked glycoprotein protruding from the viral surface. These virions were first evident at a pH below 7.0 and reached a maximum at pH 6.7 to 6.8. The spikeless virion exhibits a lower sedimentation velocity than the infectious virion, but is similar to the infectious virion in shape and size. Our present working hypothesis is that these spikeless virions are formed by proteolytic digestion of a large portion of glycoprotein after the assembly of standard virions at the cellular membrane. This is based on the following findings: (1) Electron micrographs of infected cells in which two-thirds of the progeny virions belonged to the spikeless type revealed that spikes were observed on most cell-associated virions; (2) SDS-PAGE of the spikeless virions showed a new low molecular weight polypeptide, and a polypeptide showing a similar mobility was also detected in the infectious virions in which spikes had been cleaved off by Pronase treatment; and (3) peptide mappings of both low molecular weight polypeptides revealed similar findings. The significance of the low pH of the culture medium in producing spikeless virus is under investigation.     

Intermediates in the maturation of bacteriophage lambda DNA

Intermediates in DNA maturation were studied in cells infected with temperature-sensitive mutants of phage λ. Bacteria were irradiated with UV to eliminate host DNA replication and then infected at high (nonpermissive) temperature with phage mutants in the head region of λ. Such genes have been implicated in the production of mature phage DNA. At various times after infection samples of culture were withdrawn for analysis both by viable phage assay and by sedimentation of extracted DNA in neutral and alkaline sucrose gradients. Portions of the infected cultures were also shifted down to low (permissive) temperatures and similar analyses carried out. The “rapidly sedimenting” immature DNA which accumulated under nonpermissive conditions in cells infected with mutants in genes A or E decreased under permissive conditions and at the same time material appeared in the region of mature molecules. This was not true in the case of mutants in genes B or C, where no mature molecules appeared after temperature shift-down. Assays for viable phage gave parallel results, and it was concluded that the rapidly sedimenting species observed during blocks in maturation are true intermediates.     

DNA repair-recombination functions in the DNA processing pathway of bacteriophage T4

It was previously shown that gene 49 codes for an endonuclease which functions in a terminal step in T4 maturation and that gene 49 mutations were suppressed by mutations in uv-sensitive genes, uvsX and uvsY. Here we have observed that a gene 49 mutation was also suppressed by mutations in genes 46, 47, and 59. Our results suggest that the genes uvsX, uvsY, 46, 47, and 59 function in a common DNA repair pathway. We suggest that the DNA repair functions constitute part of the DNA processing pathway and that suppression of the gene 49 mutation occurs because the DNA intermediates formed by mutational blocks in genes 46, 47 59, uvsX, and uvsY bypass the gene 49 function and reenter the DNA processing pathway just prior to the genes 16–17 function(s).     

A novel in vitro DNA packaging system demonstrating a direct role for the bacteriophage lambda FI gene product

A new in vitro bacteriophage lambda DNA packaging system is described in which all the proteins necessary for head morphogenesis are supplied by extracts of plasmid-transformed cells. This assay is used to demonstrate that the lambda FI gene product (gpFI) is necessary for maximal packaging efficiency when proheads and terminase are present in limiting amounts. A 100- to 200-fold decrease in packaging is seen when gpFI is omitted. gpFI is shown to act at and/or after the stage in packaging where proheads bind to the DNA:terminase complex.     

High-throughput sequence-based typing strategy for HLA-DRB1 based on real-time polymerase chain reaction

DNA sequencing is the gold standard for high-resolution genotyping of the highly polymorphic human leukocyte antigen (HLA) loci. In the case of hematopoietic stem cell transplantation, four-digit typing of HLA class I and II genes is indicated. We developed a group-specific, real-time polymerase chain reaction (PCR) strategy for amplification of DRB1 applying TaqMan chemistry on different real-time machines. For evaluation of genotyping accuracy and ambiguity resolution, 115 well-characterized samples were adapted. Separate analysis of each allele was possible in 100 samples (87%). Of the samples, 13% (n = 15) showed amplification in one of the eight group-specific PCR mixes: one sample according to the group DRB1*15, 16, along with 14 samples according to the group DRB1*03, *11, *13, *14. Further testing of the 14 (12.2%) samples associated with group DRB1*03, *11, *13, *14 was performed with specific intron primers. In conclusion this typing scheme generates results within 1 day, thereby making sequencing for different requirements attractive.