Integration of the bacteriophage phi 3T-coded thymidylate synthetase gene into the Bacillus subtilis chromosome

Transformation of Bacillus subtilis 168 Thy- auxotrophs with phi 3T deoxyribonucleic acid (DNA) to thymine independence was found to involve site-specific recombination of phi 3T DNA sequences with their homologous counterparts in the bacterial chromosome. During the transformation, the phage phi 3T-encoded thymidylate synthetase gene, thyP3, was shown to integrate at two genetically distinct sites in the B. Subtilis 168 chromosome. The first site was identified to be in the bacterial thymidylate synthetase gene, thyA. The second site was in a prophage (SPB) known to be carried in the host genome. The frequency of the integration of the thyP3 gene at each of the two loci and some of the parameters affecting this frequency were studied. The common origin of the thyP3 and thyA genes and their molecular evolution are also reported.     

Genomic polymorphism in the T-even bacteriophages.

We have compared the genomes of 49 bacteriophages related to T4. PCR analysis of six chromosomal regions reveals two types of local sequence variation. In four loci, we found only two alternative configurations in all the genomes that could be analyzed. In contrast, two highly polymorphic loci exhibit variations in the number, the order and the identity of the sequences present. In phage T4, both highly polymorphic loci encode internal proteins (IPs) that are encapsidated in the phage particle and injected with the viral DNA. Among the various T4-related phages, 10 different ORFs have been identified in the IP loci; their amino acid sequences have the characteristics of internal proteins. At the beginning of each of these coding sequences is a highly conserved 11 amino acid leader motif. In addition, both 5' and 3' to most of these ORFs, there is a approximately 70 bp sequence that contains a T4 early promoter sequence with an overlapping inversely repeated sequence. The homologies within these flanking sequences may mediate the recombinational shuffling of the IP sequences within the locus. A role for the new IP-like sequences in determining the phage host range is proposed since such a role has been previously demonstrated for the IP1 gene of T4.     

Distribution of bacteriophage phi 3T homologous deoxyribonucleic acid sequences in Bacillus subtilis 168, related bacteriophages, and other Bacillus species.

The Bacillus subtilis 168 chromosome was found to share extensive homology with the genome of bacteriophage phi 3T. At least three different regions of the bacterial genome hydridized to ribonucleic acid complementary to phi 3T deoxyribonucleic acid (DNA). The thymidylate synthetase gene, thyA, of B. subtilis and the sequences adjacent to it were shown to be homologous to the region in the phi 3T DNA containing the phage-encoded thymidylate synthetase gene, thyP3. SP beta, a temperate bacteriophage known to be integrated into the B. subtilis 168 chromosome, was demonstrated to be closely related to phi 3T. Other regions of the bacterial genome were also found to hybridize to the phi 3T probe. The nature and location of these sequences in the bacterial and phage chromosomes were not identified. It was shown however, that they were not homologous to either the thyP3 gene or the DNA surrounding the thyP3 gene. The chromosomes of other Bacillus species were also screened for the presence of phi 3T homologous sequences, and the thyP3 gene was localized in the linear genomes of phages phi 3T and rho 11 by heteroduplex mapping. It is suggested that the presence of sequences of phage origin in the B. subtilis 168 chromosome might contribute to the restructuring and evolution of the viral and bacterial DNAs.     

A remarkable amino acid sequence homology between a phage T4 tail fibre protein and ORF314 of phage lambda located in the tail operon

We have found that the amino acid (aa) sequence of the tip of phage T4 tail fibre (gene 37) shows more than 50% homology with the aa sequence predicted from an open reading frame (ORF314) in the phage lambda genome. ORF314 is near the 3' end of the late morphogenetic operon, beyond gene J coding for the lambda tail fibre. The homologous sequences are for the most part composed of repeated aa, the most remarkable of which is a Gly-X-His-Y-His motif where X and Y are small, uncharged aa, found six times in the T4 protein and seven times in the lambda ORF314 sequence.     

DNA sequence of genes 38 encoding a receptor-recognizing protein of bacteriophages T2, K3 and of K3 host range mutants

Genes 38, which code for a receptor-recognizing protein present at the tip of the long tail fibers, have been sequenced from phages T2, the T-even-type phage K3 and its host range mutants K3hx, K3h1 and K3h1h. The genes from phages T2 and K3 code for proteins consisting of 262 and 260 amino acid residues, respectively. Fifty amino-terminal and 25 carboxy-terminal residues are highly conserved. The amino-terminal amino acids are most likely involved in binding to the neighboring protein 37. Between residues 116 and 226 of the T2 protein and residues 116 and 223 of the K3 protein, sequences exist that are similar to sequences present in Escherichia coli outer membrane proteins and which serve as phage receptors. Most likely, all of these regions in the latter proteins are exposed on the cell surface and are part of their phage receptor areas. In the phage proteins, these sequences are flanked by stretches rich in glycine, perhaps providing an increased flexibility for the polypeptide at these sites; some "wobble" may be required during the protein 38-receptor interaction. The mutational alterations in the host range mutants were found in gene 38. In the K3hx protein, a duplication of six base-pairs caused the wild-type sequence -Gly163-Lys-Leu-Ile- to be changed to -Gly163-Lys-Leu-Lys-Leu-Ile-. In the K3h1 protein, a glutamic acid residue at position 203 was substituted by a lysine. Both alterations occurred within areas similar to outer membrane proteins. Mutant K3h1h, derived from K3h1, exhibits an extended host range as compared to K3h1. No mutational alteration, in addition to that found in K3h1, was found in g38 nor was the part of gene 37 that encodes the carboxy-terminal moiety of the protein altered. K3h1h may represent a "trigger-happy" phage. The results of this and other work show that the phage-phage receptor systems under study represent a primitive immune system.     

DNA sequence of the control region of phage D108: the N-terminal amino acid sequences of repressor and transposase are similar both in phage D108 and in its relative, phage Mu.

We have determined the DNA sequence of the control region of phage D108 up to position 1419 at the left end of the phage genome. Open reading frames for the repressor gene, ner gene, and the 5' part of the A gene (which codes for transposase) are found in the sequence. The genetic organization of this region of phage D108 is quite similar to that of phage Mu in spite of considerable divergence, both in the nucleotide sequence and in the amino acid sequences of the regulatory proteins of the two phages. The N-terminal amino acid sequences of the transposases of the two phages also share only limited homology. On the other hand, a significant amino acid sequence homology was found within each phage between the N-terminal parts of the repressor and transposase. We propose that the N-terminal domains of the repressor and transposase of each phage interact functionally in the process of making the decision between the lytic and the lysogenic mode of growth.     

Identification of Src, Fyn, Lyn, PI3K and Abl SH3 domain ligands using phage display libraries.

Many proteins involved in intracellular signal transduction contain a small, 50-60 amino acid domain, termed the Src homology 3 (SH3) domain. This domain appears to mediate critical protein-protein interactions that are involved in responses to extracellular signals. Previous studies have shown that the SH3 domains from several proteins recognize short, contiguous amino acid sequences that are rich in proline residues. While all SH3 recognition sequences identified to date share a conserved P-X-X-P motif, the sequence recognition specificity of individual SH3 domains is poorly understood. We have employed a novel modification of phage display involving biased libraries to identify peptide ligands of the Src, Fyn, Lyn, PI3K and Abl SH3 domains. With biased libraries, we probed SH3 recognition over a 12 amino acid window. The Src SH3 domain prefers the sequence XXXRPLPPLPXP, Fyn prefers XXXRPLPP(I/L)PXX, Lyn prefers RXXRPLPPLPXP, PI3K prefers RXXRPLPPLPP while the Abl SH3 domain selects phage containing the sequence PPPYPPPP(I/V)PXX. We have also analysed the binding properties of Abl and Src SH3 ligands. We find that although the phage-displayed Abl and Src SH3 ligands are proline rich, they are distinct. In surface plasmon resonance binding assays, these SH3 domains displayed highly selective binding to their cognate ligands when the sequences were displayed on the surface of the phage or as synthetic peptides. The selection of these high affinity SH3 peptide ligands provides valuable information on the recognition motifs of SH3 domains, serve as new tools to interfere with the cellular functions of SH3 domain-mediated processes and form the basis for the design of SH3-specific inhibitors of disease pathways.     

DNA sequence of the tail fiber genes 37, encoding the receptor recognizing part of the fiber, of bacteriophages T2 and K3

The DNA sequences of genes 37 of bacteriophages T2 and K3 are presented and compared with that of phage T4. The corresponding proteins constitute, as dimers, the part of the long tail fibers that recognizes the bacterial receptor. The CO2H termini of the polypeptides are located at the free ends of the fibers. Morphologically, the three phages are essentially identical, but they use different receptors. The genes from phages T4, T2 and K3 encode proteins consisting of 1026, 1341 and 1243 amino acid residues, respectively. DNA-DNA hybridizations had shown earlier that genes 37, in contrast to the gene for the major capsid protein, of a number of T-even type phages are highly polymorphic. The deduced amino acid sequences now show that this polymorphism extends to the protein primary structures. About 50 NH2-terminal residues are conserved and are probably required for binding to the adjacent protein 36. This area is followed by more or less irregularly spaced regions of non-homology, partial homology or complete homology. The heterogeneity is most prominent in a region encompassing about 600 CO2H-terminal residues of the T2 or K3 proteins. Nevertheless, the amino acid compositions of the three proteins are very similar and all are rich in glycine. It has been found that the receptor specificities of phages K3 and T2 are determined by protein 38, a polypeptide required for the efficient dimerization of protein 37 of phage T4. Proteins 38 of phages K3 and T2 are functionally interchangeable, those of T4 and T2 or K3 are not. Proteins 37 of phages K3 and T2 possess a conserved sequence of 160 CO2H-terminal residues. This area is missing in the T4 protein. This region may serve as a binding site for polypeptides 38 of phages K3 and T2. The overall picture of the protein primary structures of the three phages strongly suggests that the evolution of genes 37, which was most likely driven by selection for variations in receptor recognition specificities, has not been a steady process but has involved loss and gain of segments of DNA.     

Identification of a region of the bacteriophage T3 and T7 RNA polymerases that determines promoter specificity

Bacteriophages T7 and T3 encode DNA-dependent RNA polymerases that are 82% homologous, yet exhibit a high degree of specificity for their own promoters. A region of the RNA polymerase gene (gene 1) that is responsible for this specificity has been localized using two approaches. First, the RNA polymerase genes of recombinant T7 x T3 phage that had been generated in other laboratories in studies of phage polymerase specificity were characterized by restriction enzyme mapping. This approach localized the region that determines promoter specificity to the 3' end of the polymerase gene, corresponding to the carboxyl end of the polymerase protein distal to amino acid 623. To define more closely the region of promoter specificity, a series of hybrid T7/T3 RNA polymerase genes was constructed by in vitro manipulation of the cloned genes. The specificity of the resulting hybrid RNA polymerases in vitro and in vivo indicates that an interval of the polymerase that spans amino acids 674 to 752 (the 674 to 752 interval) contains the primary determinant of promoter preference. Within this interval, the amino acid sequences of the T3 and T7 enzymes differ at only 11 out of 79 positions. It has been shown elsewhere that specific recognition of T3 and T7 promoters depends largely upon base-pairs in the region from -10 to -12. An analysis of the preference of the hybrid RNA polymerases for synthetic T7 promoter mutants indicates that the 674 to 752 interval is involved in identifying this region of the promoter, and suggests that another domain of the polymerase (which has not yet been identified) may be involved in identifying other positions where the two consensus promoter sequences differ (most notably at position -15).     

Trinucleotide cassettes increase diversity of T7 phage-displayed peptide library

Background  

Amino acid sequence diversity is introduced into a phage-displayed peptide library by randomizing library oligonucleotide DNA. We recently evaluated the diversity of peptide libraries displayed on T7 lytic phage and M13 filamentous phage and showed that T7 phage can display a more diverse amino acid sequence repertoire due to differing processes of viral morphogenesis.     

DNA sequence heterogeneity in the genes of T-even type Escherichia coli phages encoding the receptor recognizing protein of the long tail fibers

Summary Genes (g) 36 and 37 code for the proteins of the distal half of the long tail fibers of phage T4, gene product (gp) 35 links the distal half to the proximal half of this fiber. The receptor, lipopolysaccharide, most likely is recognized by gp37. Using as probe a restriction fragment consisting of most of g36 and g37 of phage T4 the genes corresponding to g35, g36, and g37 of phages T2 and K3 (using the E. coli outer membrane proteins OmpF and OmpA, respectively, as receptors) have been cloned into plasmid pUC8. Partial DNA sequences of g37 of phage K3 have been determined. One area, corresponding to residues 157 to 210 of the 1026 residue gp37 of phage T4, codes for an identical sequence in phage K3. Another area corresponds to residues 767 to 832 of the phage T4 sequence. Amino acid residues 767 to 832 of the phage T4 sequence are almost identical in both phage proteins while the remainder is rather different. DNAs of T2, T4, T6, another T-even type phage using protein Tsx as a receptor, and 10 different T-even type phages using the OmpA protein as a receptor have been hybridized with restriction fragments covering various parts of the g37 area of phage K3. With probably only one exception all of the 13 phages tested possess unique genes 37 and within the majority of these, sequences highly homologous to parts of g37 of K3 are present in a mosaic type fashion. Other regions of these genes 37 did not show any homology with the K3 probes; in case of the OmpA specific phage M1 absence of homology was evident in most of its g37 even including the area that should serve for recognition of the cellular receptor. In sharp contrast to this situation it was found that a major part of the gene (g23) coding for the major capsid protein is rather highly conserved in all phages studied. The extreme variability in sequences existing in genes 37 might be a consequence of phages during evolution being able to more or less drastically change their receptor specifities.     

Cloning of full-length methylmalonyl-CoA mutase from a cDNA library using the polymerase chain reaction

The polymerase chain reaction was used to clone a full-length human methylmalonyl-CoA mutase cDNA from a human liver library by priming with sequences from the 5' end of a partial cDNA and sequences in the phage vector. The amino acid sequence predicted from the cDNA corresponds to the authentic amino acid sequences of peptide fragment from purified methylmalonyl-CoA mutase. The open reading frame of the cDNA encodes 742 amino acids (82,283 Da) comprising a 32 amino acid mitochondrial leader sequence and a mature protein of 710 amino acids (78,489 Da). The use of the polymerase chain reaction to "screen" the cDNA library represents a novel application of this technique. The full length will enable analysis of mutations underlying inherited methylmalonic acidemias caused by deficiency of the methylmalonyl-CoA mutase apoenzyme.     

水稻条叶枯病毒基因组组分3的克隆与序列分析

利用ＲＴ－ＰＣＲ技术,合成并扩增了水稻条叶枯病毒（ＲＳｔＶ）中国云南分离 物基因组组分３的全长ｃＤＮＡ。将ＰＣＲ产物克隆在载体ｐＣＲⅡ上,进行全序列测定。将所得核苷酸序列及其所推导的氨基酸序列与日本分离物Ｔ进行同源性比较,结果表明,在核苷酸水平上,两分离物的５’端非编码区序列相同,ｖＯＲＦ、ｖｃＯＲＦ及基因间非编码区序列的同源性分别为９７．６％、９６．８％及８７．６％,而３’端非编码区同源性为９８     

The gene for Klebsiella bacteriophage K11 RNA polymerase: sequence and comparison with the homologous genes of phages T7, T3, and SP6

Summary We determined the nucleotide sequence of gene 1 of Klebsiella phage K11, which is a member of the T7 group of phages. The largest open reading frame corresponds to a polypeptide with 906 amino acids and a molecular weight of 100383 daltons. The deduced amino acid sequence of this polypeptide shows 71% homology to the T7 RNA polymerase (the product of T7 gene 1), 72% homology to the T3 RNA polymerase and 27% homology to the SP6 RNA polymerase. Divergent evolution was clearly most pronounced in the amino-terminal portion.     

Identification of trimannoside-recognizing peptide sequences from a T7 phage display screen using a QCM device

Here, we report on the identification of trimannoside-recognizing peptide sequences from a T7 phage display screen using a quartz-crystal microbalance (QCM) device. A trimannoside derivative that can form a self-assembled monolayer (SAM) was synthesized and used for immobilization on the gold electrode surface of a QCM sensor chip. After six sets of one-cycle affinity selection, T7 phage particles displaying PSVGLFTH (8-mer) and SVGLGLGFSTVNCF (14-mer) were found to be enriched at a rate of 17/44, 9/44, respectively, suggesting that these peptides specifically recognize trimannoside. Binding checks using the respective single T7 phage and synthetic peptide also confirmed the specific binding of these sequences to the trimannoside-SAM. Subsequent analysis revealed that these sequences correspond to part of the primary amino acid sequence found in many mannose- or hexose-related proteins. Taken together, these results demonstrate the effectiveness of our T7 phage display environment for affinity selection of binding peptides. We anticipate this screening result will also be extremely useful in the development of inhibitors or drug delivery systems targeting polysaccharides as well as further investigations into the function of carbohydrates in vivo.