The construction of the hybrid plasmid containing genes of the central part of phage T4 baseplate and gene 29 product separation

A fragment of E. coli bacteriophage T4 genome including the four genes (genes 51, 27, 28, 29) coding for the central plug proteins was cloned into plasmid pMCC17. The genes present on this fragment were expressed in E. coli in the absence of phage infection producing hub proteins, which could be identified on polyacrylamide gels. By applying affinity chromatography protein 29 was purified from extracts of E. coli transformed with this hybrid plasmid. The isolated protein had the ability to complement T4 29 amber mutants. The molecular weight of the purified protein was estimated as 75,000 to 85,000 depending on the composition of SDS-polyacrylamide gel used for the assay.     

Construction and characterization of hybrids containing genes coding for proteins of the central part of bacteriophage T4 baseplate.

The central part (hub or plug) of bacteriophage T4 baseplate consist of several proteins which are present in only few copies per phage particle. The presence of these minor baseplate components was inferred from the genetic data but only some of them were identified as distinct proteins species by biochemical analysis. We have constructed a number of plasmids containing segments of bacteriophage T4 genome coding for baseplate proteins. The following genes were cloned into expression vectors: 54, 48, 29, 28, 27, 51, 26 and 25. The presence of a particular gene product was confirmed by in vivo complementation test. On the basis of these results we could more precisely localize the position of a particular gene on T4 phage genetic map. The hybrids contain sets of genes which make aggregation impossible, so bacteria harbouring these plasmids are convenient starting point for the purification of baseplate proteins.     

A DNA fragment containing the groE genes can suppress mutations in the Escherichia coli dnaA gene

Summary An 8.2 kb fragment of E. coli chromosomal DNA, when cloned in increased copy number, suppresses the dnaA46 mutation, and an abundant protein of about 68 kd (60 kd when measured by us), encoded by the fragment, is essential for the suppression (Takeda and Hirota 1982). Mapping experiments show that the fragment originates from the 94 min region of the chromosome. It encodes several proteins but only one abundant polypeptide of the correct size, the product of the groEL gene. Suppression by the fragment is allele specific; those mutations which map to the centre of the gene are suppressed. Other initiation mutants including dnaA203, dnaA204, dnaA508, dnaAam, dnaC, dnaP and dnaB252 are not suppressed. Most suppressed strains are cold-sensitive suggesting an interaction between the mutant proteins (or their genes) and the suppressing protein or proteins.     

T4 DNA replication and viral gene expression

The normal dependence of “late” T4 gene expression on concurrent viral DNA replication is circumvented in cells infected with a triple mutant in which viral DNA polymerase, DNA ligase, and the exonuclease functions of genes 46 or 47 are defective. Acrylamide gel electrophoresis of labeled proteins from infected cells has made possible an extension of the analysis of replication-uncoupled T4 protein synthesis. We find a number of late T4 proteins synthesized: the products of genes 34, 37, 18, 23 and 24. Processing of the gene 23 product, normally headassembly dependent, occurs, but with considerably diminished efficiency compared to wild-type infection. Late T4 protein synthesis in replication-uncoupled infection retains a requirement for T4 gene 33 and gene 55 function. Finally, a number of “early” T4 gene products, normally shut off late in wildtype infection, continue to be synthesized late in replication-uncoupled infection, concurrently with the late proteins.     

Structure and location of gene product 8 in the bacteriophage T4 baseplate

Many bacteriophages, such as T4, T7, RB49, and phi29, have complex, sometimes multilayered, tails that facilitate an almost 100% success rate for the viral particles to infect host cells. In bacteriophage T4, there is a baseplate, which is a multiprotein assembly, at the distal end of the contractile tail. The baseplate communicates to the tail that the phage fibers have attached to the host cell, thereby initiating the infection process. Gene product 8 (gp8), whose amino acid sequence consists of 334 residues, is one of at least 16 different structural proteins that constitute the T4 baseplate and is the sixth baseplate protein whose structure has been determined. A 2.0A resolution X-ray structure of gp8 shows that the two-domain protein forms a dimer, in which each monomer consists of a three-layered beta-sandwich with two loops, each containing an alpha-helix at the opposite sides of the sandwich. The crystals of gp8 were produced in the presence of concentrated chloride and bromide ions, resulting in at least 11 halide-binding sites per monomer. Five halide sites, situated at the N termini of alpha-helices, have a protein environment observed in other halide-containing protein crystal structures. The computer programs EMfit and SITUS were used to determine the positions of six gp8 dimers within the 12A resolution cryo-electron microscopy image reconstruction of the baseplate-tail tube complex. The gp8 dimers were found to be located in the upper part of the baseplate outer rim. About 20% of the gp8 surface is involved in contacts with other baseplate proteins, presumed to be gp6, gp7, and gp10. With the structure determination of gp8, a total of 53% of the volume of the baseplate has now been interpreted in terms of its atomic structure.     

Cloning of the Bacillus subtilis DNA fragment containing the genes for lysine and riboflavin biosynthesis

The SalI fragment of chromosomal DNA of Bacillus subtilis carrying the gene for lysine biosynthesis and the regulatory operator region (ribO) from the riboflavin gene was cloned into Escherichia coli cells. This fragment was shown to contain the gene coding for lysine synthesizing enzyme. Localization of this gene in Bac. subtili was determined. New plasmids pLRS33 and pLRB4 were constructed using pBR322; they carry a fragment homologous to pLP102 plasmid containing the operon for riboflavin biosynthesis.     

Nucleotide sequence of a Xenopus laevis mitochondrial DNA fragment containing the D-loop, flanking tRNA genes and the apocytochrome b gene

Extensive corrections of the nucleotide sequence of the Xenopus laevis mitochondrial (mt) displacement (D) loop and surrounding genes [Wong et al., Nucl. Acids Res. 11 (1983) 4977-4995] are reported, including addition of two stretches of nucleotides and 60 scattered modifications. The additional sequences presented here correspond to the apocytochrome b gene, the tRNAGlu gene and part of URF6. This allows us to propose a conformational model for the X. laevis apocytochrome b protein and also permits comparisons with mammalian mtDNA. The D-loop sequence is poorly conserved except for sequences involved in the regulation of the mt genome (conserved sequence blocks and the DNA polymerase stop sequences). On the other hand, all genes show marked conservation both of their nucleotide sequence and their respective location on the mt genome. Organization of the genetic information described for mammalian mtDNA also holds for the X. laevis mtDNA. This result strongly suggests that all animal vertebrate mtDNAs have followed the same evolutionary pathway.     

Bacteriophage T4 tail assembly: proteins of the sheath, core and baseplate

Structural intermediates in phage tail formation have been isolated by sucrose gradient centrifugation from cells infected with mutants blocked at various stages in tail assembly. The polypeptide chains of these structures containing ¹⁴C-labeled amino acids have been analyzed by sodium dodecyl sulfate—acrylamide gel electrophoresis, enabling us to identify the proteins forming the various morphological components of the tail. Comparison of sheathed tails with corebaseplates shows that the contractile sheath is composed of a single species of subunit, the product of gene 18 (mol.wt 80,000). The site for head attachment terminating the tail is composed of the product of gene 15 (mol.wt 35,000). Comparison of core-baseplates with free baseplates shows that the tail core is composed of a single species of subunit, the product of gene 19 (mol.wt 21,000).Free baseplates are composed of at least twelve species of proteins: the products of genes 6, 7, 8, 9, 10, 11, 12 and 29, and four genetically unidentified species.The incomplete tails which accumulate in cells infected with mutants defective in genes 9, 11 and 12, which specify proteins on the outside of the baseplate, have also been characterized. Tails from 9^? lysates lack only P9. Tails from 11^? lysates lack both Pll and P12. Tails from 12^? infection lack only P12. Incorporation of P12 into the baseplate requires the function of gene 57, which is also required for tail fiber assembly. P57 thus appears to take part in the maturation of three different phage structural proteins.The sequential nature of the protein interactions in tail formation is discussed in terms of the regulation of morphogenesis at the level of assembly.     

Somatic cell mapping and restriction fragment analysis of bovine genes for fibronectin and gamma crystallin

DNAs from cow-hamster and cow-mouse somatic hybrid cells segregating bovine chromosomes have been analyzed by Southern blotting and hybridization with human fibronectin and gamma crystallin probes. Concordancy of retention of these bovine genes was compared to cattle isozyme loci representing previously described syntenic groups. Bovine fibronectin (FNI) and gamma crystallin (CRYG) fragments were concordant with each other and with isocitrate dehydrogenase 1 (IDH1), representing the bovine syntenic group U17. The syntenic relationship of these genes is conserved on human chromosome 2q and also on mouse chromosome 1. In addition, bovine RFLPs were identified with both fibronectin and gamma crystallin probes. These polymorphisms will be used to study recombination between the syntenic loci in pedigreed herds and to mark a segment of the bovine genome that is likely homologous to the Lsh region of mouse chromosome 1, which confers resistance in mice to several intracellular parasites.     

Biomolecular response of oxanine in DNA strands to T4 polynucleotide kinase, T4 DNA ligase, and restriction enzymes

Oxanine (Oxa), generated from guanine (Gua) by NO- or HNO₂-induced nitrosative oxidation, has been thought to cause mutagenic problems in cellular systems. In this study, the response of Oxa to different enzymatic functions was explored to understand how similarly it can participate in biomolecular reactions compared to the natural base, Gua. The phosphorylation efficiency of the T4 polynucleotide kinase was highest when Oxa was located on the 5′-end of single stranded DNAs compared to when other nucleobases were in this position. The order of phosphorylation efficiency was as follows; Oxa > Gua > adenine (Ade) ∼ thymine (Thy) > cytosine (Cyt). Base-pairing of Oxa and Cyt (Oxa:Cyt) between the ligation fragment and template was found to influence the ligation performance of the T4 DNA ligase to a lesser degree compared to Gua:Cyt. In addition, EcoRI and BglII showed higher cleavage activities on DNA substrates containing Oxa:Cyt than those containing Gua:Cyt, while BamHI, HindIII and EcoRV showed lower cleavage activity; however, this decrease in activity was relatively small.     

DNA methylation and chromatin structure regulate T cell perforin gene expression

Perforin is a cytotoxic effector molecule expressed in NK cells and a subset of T cells. The mechanisms regulating its expression are incompletely understood. We observed that DNA methylation inhibition could increase perforin expression in T cells, so we examined the methylation pattern and chromatin structure of the human perforin promoter and upstream enhancer in primary CD4(+) and CD8(+) T cells as well as in an NK cell line that expresses perforin, compared with fibroblasts, which do not express perforin. The entire region was nearly completely unmethylated in the NK cell line and largely methylated in fibroblasts. In contrast, only the core promoter was constitutively unmethylated in primary CD4(+) and CD8(+) cells, and expression was associated with hypomethylation of an area residing between the upstream enhancer at -1 kb and the distal promoter at -0.3 kb. Treating T cells with the DNA methyltransferase inhibitor 5-azacytidine selectively demethylated this area and increased perforin expression. Selective methylation of this region suppressed promoter function in transfection assays. Finally, perforin expression and hypomethylation were associated with localized sensitivity of the 5' flank to DNase I digestion, indicating an accessible configuration. These results indicate that DNA methylation and chromatin structure participate in the regulation of perforin expression in T cells.     

Enzymatic synthesis, ligation, and restriction of DNA containing deoxy-4-thiothymidine.

Phage fd RF I DNA1 about 90% substituted by deoxy-4-thiothymidine (s4Td) in the codogenic strand was synthesized by the simultaneous actions of DNA polymerase I and DNA ligase. While the rate of DNA synthesis was considerably reduced, the yield the rate of DNA synthesis was considerably reduced, the yield was not affected in the presence of s4TdTP. The conversion of RF II to RF I DNA by DNA ligase was even improved. This effect seems to be related with an altered ratio of affinity of polymerase and ligase for the s4Td-containing substrate. The presence of the base analogue in the DNA was verified independently by chromatographic and spectroscopic methods. The modified genome could be cleaved by restriction endonucleases Hpa II (C/CGG)d and Taq I (T/CGA)d. A number of the fragments produced showed altered mobilities under the conditions of polyacrylamide gel electrophoresis.     

A nomenclature for restriction enzymes,DNA methyltransferases,homing endonucleases and their genes

A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different Type II enzymes now identified and provides a system for naming the putative genes found by sequence analysis of microbial genomes.     

DNA restriction fragment analysis of the proopiomelanocortin gene in schizophrenia and bipolar disorders.

The method of DNA restriction fragment analysis using gene probes for the proopiomelanocortin (POMC) gene was employed to detect possible molecular variation in the POMC gene in schizophrenia and bipolar illness. No gross structural abnormalities in restriction fragments were observed with the set of restriction enzymes used. Two allelic restriction sites were observed giving rise to fragment length polymorphisms. One of these is a new polymorphism, not previously reported, which will be of value as a linkage marker. The associations between the two DNA polymorphisms that are closely linked to the POMC gene and both schizophrenia and bipolar disorder were investigated. No association was found, thus adding weight to the evidence that there are no alterations in the POMC gene in schizophrenia and bipolar illness.     

Isolation and characterization of precursors in T4 baseplate assembly the complex of gene 10 and gene 11 products

The first multi-protein precursor in the assembly of the radial arms of the T4 baseplate has been purified to homogeneity. The complex was isolated from cells infected with a mutant blocked in the subsequent step in baseplate arm assembly. The assay for this precursor exploited the fact that the complex contains the target antigen of the neutralizing antibodies found in antibaseplate serum (Berget & King, 1978).The complex is composed of gene 10 protein (M_r, 88,000) and gene 11 protein (M_r, 24,000). Analytical ultracentrifugation experiments revealed a molecular weight of 258,000 and a sedimentation coefficient of 9.3 S for the complex. The overall and single polypeptide chain molecular weights are consistent with the complex containing two gene 10 polypeptides and four gene 11 polypeptides. Visualization of the complex in the electron microscope revealed an asymmetric angular structure. The shape, together with the previous identification of gene 11 product as the tail-spike protein (Crowther et al., 1977), indicates that the complex forms the body of the spikes and vertices of the hexagonal baseplate.Using an in vitro baseplate assembly assay, it was possible to demonstrate that the complex contains both the assembly-active gene 10 and gene 11 products. Gene 11 product (from 10^? extracts) can convert 11^? particles to viable phage. However, the complex lacked this activity, indicating that it does not readily dissociate. The precursor complex could be dissociated with denaturing solvents. Upon returning to physiological conditions, both the antigenic and biological activities of the gene 11 product could be recovered. The biological activity of the gene 10 product was not regained.