Genetic analysis of the cloned genome of phage Mu

Randomly sheared fragments of Mu cts62 DNA have been cloned into the EcoRI cleavage site of plasmid p0P203(UV-5)-3, a derivative of pMB9 containing the lactose operator promoter, using poly (dA · dT) tailing with terminal transferase. The extent of Mu DNA carried by the recombinant plasmids was determined genetically by crosses with known markers in essential genes of Mu in rec⁺ backgrounds. Plasmids which rescued essential genes were transformed to rec^? backgrounds to check for expression of the Mu genes. Plasmids were found which rescued and complemented 23 of the 24 essential genes of Mu. Many of these plasmids carried overlapping segments of the Mu genome. The plasmid library was also screened for clones containing the nonessential genes gin and mom. Clones carrying gene A were not found in the initial screening, however, several clones which were resistant to infection by Mu due to expression of the Mu repressor were able to rescue and complement a D108cts-MuAts hybrid phage at 43°.     

Partial correlation of the genetic and physical maps of bacteriophage Mu

λpMu phage particles that carry varying amounts of the Mu genome have been used to correlate the genetic and physical maps of phage Mu DNA. Two complementary techniques, restriction enzyme fragment analysis and electron microscope heteroduplex mapping, have been employed to identify the segments of Mu DNA present in the λpMu particles whose genetic complement had been determined by Mu amber mutant marker rescue. These analyses permitted us to assign a physical location of genes A to lys on one end (immunity end) of the Mu genome, and genes L to S on the other end (variable end). None of the hybrids described here included genes E to K which lie in the middle part of Mu DNA.     

铜绿假单胞菌噬菌体PaP3全基因组在感染宿主菌PA3过程中的分时期表达研究

目的明确铜绿假单胞菌噬菌体PaP3在感染宿主菌PA3后其全基因组的分时期表达模式。方法利用基因芯片检测噬菌体PaP3在感染宿主菌PA3后不同时间(5 min,10 min,20 min,30 min,80 min)其全基因组(含71个预测的ORF)在转录水平的表达谱变化,并进行非监督层次聚类(hierarchical clustering)分析。利用蛋白质合成抑制剂氯霉素(Cm)和DNA复制抑制剂磷酸乙酸(PAA)实验确定PaP3全基因组的分时期表达情况。结果根据时间表达模式的不同,PaP3全基因组可分为3大类:15个早期基因(ORF 71-57)、35个中期基因(ORF 56-22)及21个晚期基因(ORF 21-1)。在PaP3基因组结构中,这3类基因各自串联分布且可能受不同的操纵子调控。结论 PaP3感染过程中其早期、中期及晚期基因随时间有序表达,为深入了解噬菌体基因表达模式及生命复制周期奠定基础,并为了解噬菌体与宿主及机体免疫系统的的相互作用提供了基础。     

Sequence requirements for translation initiation of Rhopalosiphum padi virus ORF2

Rhopalosiphum padi virus (RhPV) is an aphid-infecting virus with a 10-kb ssRNA genome that contains two large open reading frames (ORFs). ORF1 and ORF2 encode the nonstructural and structural polyproteins, respectively. Both ORFs are preceded by noncoding regions of 500 nt that could function as internal ribosome entry segments (IRESes). The sequence for ORF2 lacks an obvious initiation codon, but an out-of-frame AUG codon is present that could translate ORF2 through a +1 frameshift. To investigate the mechanisms of translation initiation of ORF2, a series of point and deletion mutations were constructed and transcribed and translated in vitro. A bicistronic plasmid containing two copies of the RhPV intergenic region translated both ORFs efficiently, indicating that the region functioned as an IRES in vitro. Deletion analysis showed that the region required for activity of the IRES extended from 178 nt upstream and 6 nt downstream of the 5' border of ORF2. Changes in the out-of-frame AUG codon had little effect on translation initiation, but mutations that included the first and second codons of ORF2 or that disrupted a putative pseudoknot structure near the 3' end of the IRES failed to initiate protein synthesis. Sequence analysis of the in vitro synthesized proteins showed that the first amino acid of the polyprotein corresponded to the second codon of ORF2. These results show that in vitro the noncoding region upstream of RhPV ORF2 functions as an IRES that contains a pseudoknot that directs translation initiation at a non-AUG codon. If the in vitro synthesized proteins have not been processed by an aminopeptidase, translation is initiated at the second (GCA) codon of ORF2.     

The genome of bacteriophage phiKMV,a T7-like virus infecting Pseudomonas aeruginosa

The complete DNA sequence of a new lytic T7-like bacteriophage phiKMV is presented. It is the first genome sequence of a member of the Podoviridae that infects Pseudomonas aeruginosa. The linear G + C-rich (62.3%) double-stranded DNA genome of 42,519 bp has direct terminal repeats of 414 bp and contains 48 open reading frames that are all transcribed from the same strand. Despite absence of homology at the DNA level, 11 of the 48 phiKMV-encoded putative proteins show sequence similarity to known T7-type phage proteins. Eighteen open reading frame products have been assigned, including an RNA polymerase, proteins involved in DNA replication, as well as structural, phage maturation, and lysis proteins. Surprisingly, the major capsid protein completely lacks sequence homology to any known protein. Also, the strong virulence toward many clinical P. aeruginosa isolates and a short replication time make phiKMV attractive for phage therapy or a potential source for antimicrobial proteins.     

Analysis of the complete genome of a virus associated with twisted leaf disease of cherry reveals evidence of a close relationship to unassigned viruses in the family Betaflexiviridae

The genome of a virus associated with cherry twisted leaf disease (CTLaV, isolate ZH) was sequenced and consists of 8431 nucleotides, excluding a poly(A) tail at the 3′ end. Genome analysis shows that CTLaV-ZH represents a new and distinct species and has a genome organization similar to those of unassigned viruses in the family Betaflexiviridae. The CTLaV-ZH genome has five open reading frames (ORFs), with putative ORFs within ORF2 and ORF5, identified as ORF2a and ORF5a, respectively. The AUG start codons of ORF2a and ORF5a are in contexts suitable for efficient translation, with appropriate stop codons in frame.     

Linkage of the variable end of the bacteriophage Mu DNA to the tail

Bacteriophage Mu particles were disrupted by treatment at high pH, and the DNA released from the phage was analyzed by electron microscopy. The DNA appeared to be associated with the proximal end of the tail. Denaturation maps of Mu DNA were obtained by heating and ultraviolet irradiation. The profiles of the two maps were comparable and indicated that the same end of the Mu genome was always connected to the sheath structure. Analysis of the DNA fragments after treatment of the DNA-tail complex with the endonuclease Eco R1 showed that the variable end of the Mu DNA was attached to the tail.     

Mapping of a site for packaging of bacteriophage Mu DNA

Mini-Mu containing variable DNA sequences at the left end, were tested for their ability to be packaged by a helper Mu phage. It was shown that a packaging site of Mu is situated between nucleotides 35 and 58 of the left end.     

Augmentation of the antimicrobial efficacy of antibiotics by filamentous phage

A significant increase in sensitivity to several antibiotics was observed in vitro after infection of the two Pseudomonas aeruginosa strains O1 and K with the filamentous phage Pf3 and Pf1, respectively. Moreover, upon infection with phage Pf1 a P. aeruginosa K strain harboring a plasmid-borne gentamicin resistance gene could be resensitized to the antibiotic. We further show that BALB/c mice were rescued from lethal infections with P. aeruginosa K by concomitant treatment with phage Pf1 and low concentrations of gentamicin, neither of which was able to cure the infection when administered alone.     

The amino terminus of the bacteriophage D108 transposase protein contains a two-component sequence-specific DNA-binding domain

We have cloned various amino-terminal domains of the transposable bacteriophage D108 A protein (transposase) into a high-copy-number expression vector and visualized the D108 polypeptides and fusion proteins expressed by the recombinant plasmids. By using crude protein extracts made from strains harboring these recombinant plasmids, we have performed band competition assays and DNasel footprinting on a 32P-end-labeled DNA restriction fragment which contained the Mu right end (to which the Mu A protein binds) and have shown that these fusion proteins in crude extracts can specifically bind to this DNA substrate. Furthermore, we have divided the amino-terminal 13 kDa of the D108 A protein (which may contain two bi-alpha-helical protein structures) in half and have shown that each half is capable of independent binding to the Mu attR site. These results suggest that the D108 transposase protein contains multiple DNA-binding domains which may be required for the complex protein-DNA interactions of D108 transposition.     

Location of the “variable end” of Mu DNA within the bacteriophage particle

When bacteriophage Mu is subjected to mild formaldehyde cross-linking conditions and spread for electron microscopy, the phage heads lyse and one end of the released DNA is found attached to the proximal end of the phage tail. If spreading is performed at pH 11.0–11.2 the DNA exhibits regions of partial denaturation in characteristic locations along the DNA molecule. Examination of the partial denaturation patterns of Mu DNA-tail complexes for wild-type Mu and for the insertion mutant MuX reveals that the tail is attached to a unique end of the DNA. Comparison of denaturation maps of DNA-tail complexes with those of Mu DNA heteroduplexes containing G bubbles and split ends indicates that the tail is attached to the variable end of Mu DNA. Similar analysis of denaturation maps of Mu DNA partially ejected through the tail demonstrates that the variable end is also the first end to be ejected from the phage particle. These results support the hypothesis that the variable end is the last section of Mu DNA to be packaged and the first to be released upon injection.     

Replication of mini-Mu prophage DNA

Thermoinducible mini-Mu prophage which have internal deletions spanning various portions of the αG regions were used to analyze the replication process of bacteriophage Mu. Two mini-Mu's, Mu Δ26 and Mu Δ123, containing at least the repressor gene c, the replication genes A and B, and both termini were shown to replicate upon induction even though most of the internal region of the phage DNA was deleted. Mini-Mu (A⁺B^?), deleted for part of the B gene, did not replicate upon induction; complementation with nonreplicating left end Mu fragments supplying B gene product resulted in a very low level of Mu replication, whereas fragments supplying gene B and additional functional DNA between genes B and C allowed extensive replication of the mini-Mu DNA. These results indicate that in the region between genes B and C a function(s) exists which amplifies the replication of Mu DNA.     

Sequence homology and absence of mRNA defines a possible pseudogene member of the Trypanosoma cruzi gp85/sialidase multigene family.

A genomic clone, pTt21, containing DNA apparently transcribed specifically in Trypanosoma cruzi trypomastigotes, was obtained by differentially screening a genomic library with trypomastigote and epimastigote cDNA. This 3444-bp clone contained open reading frames at each end, separated by a 1.8-kb non-coding region. The translated polypeptide from the 3' open reading frame (ORF2) of 1037 bp had 25-30% identity with 5 recently published T. cruzi gp85/sialidase sequences, and 20-25% identity with bacterial sialidases. Rabbit antiserum raised against an Escherichia coli fusion protein derived from the 5' open reading frame (ORF1) identified a surface antigen of 160 kDa, specifically expressed in trypomastigotes. A probe containing the first 211 bp from ORF1 was used to obtain a complete copy (c1821) of a gene that was closely related to ORF1, and encoded another member of the gp85/sialidase family. c1821 encodes a protein of 897 amino acids, but assignment of the N-terminus of the polypeptide was not possible. The 5'-most start codon is an unfavourable context to act as a translation initiator, it does not align with the initiator methionines of other gp85/sialidase sequences, nor is it followed by a signal peptide sequence characteristically found in other gp85/sialidase sequences. Although homology with the 5' ends of other gp85/sialidase sequences decays towards the 5' end of c1821, alignment of c1821 with 4 other gp85/sialidases indicated that the coding sequence should extend upstream at least 160 amino acids. In this region of c1821 there are multiple stop codons in each frame. The presence of the stop codons, the alignment data and our inability to amplify reverse transcribed mRNA using four internal primers, suggest that c1821 may not be present as a mature mRNA and is a pseudogene. Comparison of the apparently non-repetitive 3' coding domain of c1821 with the corresponding repetitive domains of two other members of the gp85/sialidase family revealed a high degree of similarity in nucleotide but not in amino acid sequence, and c1821 may thus represent an evolutionary intermediate between sub-families of the gp85/sialidase superfamily.