Replication of bacteriophage f1: A complex containing gene II protein in gene V mutant-infected bacteria

A dense complex has been isolated from bacteria infected with gene V amber mutant f 1 bacteriophage. The major protein in this complex is the f 1 bacteriophage-specific gene II protein. Other proteins in the complex include the f 1 bacteriophage coat protein and proteins which migrate on sodium dodecyl sulfate/polyacrylamide gel electrophoresis with the f1 bacteriophage-specific gene III, gene IV and X protein. A protein of approximately 20,000 M_r is also present in the complex. Examination of bacteria infected with gene V mutant f1 bacteriophage revealed the complex as a densely staining amorphous body which appears to be associated with the cytoplasmic membrane. Bacteria infected with f1 bacteriophage that contain amber mutations in genes other than gene V do not contain this complex.     

In-frame overlapping genes: the challenges for regulating gene expression

In-frame overlapping genes in phage, plasmid and bacterial genomes permit synthesis of more than one form of protein from the same gene. Having one gene entirely within another rather than two separate genes presumably precludes recombination events between the identical sequences. However, studies of such gene pairs indicate that the overlapping arrangement can make regulation of the genes more difficult. Here, we extend studies of in-frame overlapping genes II and X from filamentous phage f1 to determine if translational controls are required to regulate the gene properly. These genes encode proteins (pII and pX) with essential but opposing roles in phage DNA replication. They must be tightly regulated to maintain production of the proteins at relative steady state levels that permit continuous replication without killing the host. To determine why little or no pX appears to be made on the gene II/X mRNA, gene II translation was lowered by progressively deleting into the gene II initiator region. Increased pX translation resulted, suggesting that elongating ribosomes on the gene II mRNA interfere with internal initiation on the gene X ribosome binding site and limit gene X translation. As judged from systematically lowering the efficiency of suppression at a gene II amber codon upstream from the gene X start, the already modest level of gene II translation would have to be reduced by more than twofold to relieve all interference with internal initiation. Further downregulation of gene X expression proved to be required to maintain pX at levels relative to pII that are tolerated by the cell. Site-directed mutagenesis and nuclease mapping revealed that the gene X initiation site is sequestered in an extended RNA secondary structure that lowers gene X translation on the two mRNAs encoding it. The more general implications of the results for expression of in-frame overlapping genes are discussed.     

Regulation of the Escherichia coli hfq gene encoding the host factor for phage Q beta.

The host factor (HF-I) for phage Q beta RNA replication is a small protein of 102 amino acid residues encoded by the hfq gene at 94.8 min on the Escherichia coli chromosome. The synthesis rate of HF-I at the exponential-growth phase is higher than at the stationary phase, and it increases concomitantly with the increase in cell growth rate. The intracellular level of HF-I is about 30,000 to 60,000 molecules per cell, the majority being associated with ribosomes as one of the salt wash proteins. Taken together, we suggest that HF-I is one of the growth-related proteins.     

Separation and quantitation of intracellular forms of poliovirus RNA by agarose gel electrophoresis.

Intracellular poliovirus-specific RNA species can be measured directly by electrophoresis of total cytoplasmic nucleic acids through 1% agarose gels, resulting in the separation of single- and double-stranded forms of poliovirus RNA from each other and from HeLa cell 28S ribosomal RNA. Single-stranded RNA molecules differing by only 15% in length are resolved in this gel system. RNA species can be visualized as fluorescen bands appearing after staining of the gels with ethidium bromide and observation under ultraviolet illumination. The total amount of RNA can be determined by densitometric quantitation of the fluorescent response. In this way, the amount of poliovirus-specific RNA within the cytoplasm of HeLa cells infected for various times has been estimated. At 170-min postinfection, there are 0.67 X 10(5) molecules of single-stranded poliovirus RNA per cell and at 230 min, the amount has increased to 3.7 X 10(5) molecules/cell. Poliovirus double-strnaded RNA reaches a maximum of 0.7 X 10(5) molecules/cell at 330 min after infection.     

Bacteriophage f1 DNA replication genes. II. The roles of gene V protein and gene II protein in complementary strand synthesis

Filamentous phage gene V, which encodes a single-stranded DNA binding protein, has been cloned and placed under control of the lac promoter. Cells bearing the clone are refractory to filamentous phage infection if the expression of the gene is induced with isopropyl-1-thio-beta-D-galactoside. The inhibition of infection is shown to occur at an early stage, and can be reversed if the cells express gene II in addition to gene V protein. These observations support the hypothesis that gene II protein, in addition to its role in nicking and facilitating the synthesis of phage viral (+) strand DNA, functions to prevent the gene V-mediated inhibition of complementary (-) strand synthesis. We proposed a model in which the absolute and relative concentrations of the products of genes II, X and V determine whether a single strand is to be exported as phage or incorporated into double-stranded replicative form DNA.     

Molecular requirements for the mu-induced light chain gene rearrangement in pre-B cells.

During B cell differentiation rearrangement of immunoglobulin (Ig) genes is partially regulated by the Ig proteins. Rearrangement of heavy (H) chain genes is inhibited, whilst that of light (L) chain genes is induced by the membrane form of the mu H chain. In order to analyse additional structural requirements of mu induced L chain gene rearrangement we transfected wild-type mu and mutant mu constructs lacking functional exons encoding the first or second constant domains into Abelson murine leukemia virus (AMuLV) transformed pre-B cells. All mu chains are expressed on the surface of the pre-B cell and all associate with omega and iota, two proteins forming a surrogate light chain, necessary for mu membrane expression. Nevertheless, only wild-type mu and not the mutant mu proteins promote L gene rearrangement. A heterodimer of proteins with Mr of 33 kd and 36 kd was found associated with wild-type but not with the mutant mu proteins. Continuous presence of mu is required for L chain gene recombination since loss of mu stopped and readdition of mu started L gene rearrangement. We propose that the protein complex composed of mu and the 33 kd/36 kd protein heterodimer is responsible for the activation of the L chain gene locus and its rearrangement.     

Structure and flanking regions of soybean seed protein genes

We have characterized the structure and flanking region of genes representing two, coordinately expressed, soybean seed protein gene families. One family directs the synthesis of the major storage protean glycinin; the other encodes a 15.5 kd polypeptide of unknown function. DNA blot hybridization experiments showed approximately three, nonallelic genes in the glycinin family and two in the 15 kd protein family, and showed that these families are not selectively amplified or rearranged during embryogeny. R-loop and S1 nuclease mapping studies demonstrated no detectable introns in the 15 kd protein genes but at least one and possibly two in the glycinin genes. No interfamily clustering of these genes occurs within a 10-15 kb chromosomal domain. Nor are they contiguous to other genes expressed at moderate levels during embryogenesis. Each of them, however, is contiguous to a gene expressed at another developmental period in the leaf. These leaf genes encode rare class messages which constitute only 1 X 10(-5%) of the leaf mRNA, or about one molecule per cell. R-loop analysis of two leaf genes showed that one contains no detectable introns while the other possesses at least three. DNA gel blot studies showed that only one of the seed protein genomic clones contains an interspersed repetitive DNA element. Pairwise cross-hybridization studies did not detect any flanking sequences shared by the 15 kd protein, glycinin and leaf genes.     

Selection and characterization of randomly produced mutants of gene V protein of bacteriophage M13.

Gene V protein of bacteriophage Ff (M13, f1, fd) is a master regulator of phage DNA replication and phage mRNA translation. It exerts these two functions by binding to single-stranded viral DNA or to specific sequences in the 5' ends of its target mRNAs, respectively. To study the structure/function relationship of gene V protein, M13 gene V was inserted in a phagemid expression vector and a library of missense and nonsense mutants was constructed by random chemical mutagenesis. Phagemids encoding gene V proteins with decreased biological activities were selected and the nucleotide sequences of their gene V fragments were determined. Furthermore, the mutant proteins were characterized both with respect to their ability to inhibit the production of phagemid DNA transducing particles and their ability to repress the translation of a chimeric lacZ reporter gene whose expression is controlled by the promoter and translational initiation signals of M13 gene II. From the data obtained, it can be deduced that the mechanism by which gene V protein binds to single-stranded DNA differs from the mechanism by which it binds to its target sequence in the gene II mRNA.     

The pim-1 oncogene encodes two related protein-serine/threonine kinases by alternative initiation at AUG and CUG.

The pim-1 gene is frequently found activated by proviral insertion in murine T cell lymphomas. Overexpression of pim-1 in lymphoid cells by transgenesis formally proved its oncogenic potential. The pim-1 cDNA sequence predicts that both murine and human pim-1 encode a 34 kd protein with homology to protein kinases. In this study, we show that the murine pim-1 gene encodes a 44 kd protein in addition to the predicted 34 kd protein. The 44 kd protein is an amino-terminal extension of the 34 kd protein and is synthesized by alternative translation initiation at an upstream CUG codon. Contrary to previous findings by others, we provide evidence that both murine and human pim-1 gene products are protein-serine/threonine kinases. Murine 44 kd and 34 kd pim-1 proteins exhibit comparable in vitro kinase activity and are both mainly cytoplasmic, but they differ in in vivo association state and half-life.     

Identification of the dnaA and dnaN gene products of Escherichia coli

Summary A specialized transducing lambda phage carrying the dnaN and dnaA genes of Escherichia coli specifies two proteins of about 41 and 48 kilodaltons (kd). The temperature-sensitive mutations, dnaN59 and dnaA167, were found to result in altered isoelectric points of the 41 and 48 kd proteins, respectively. Thus the dnaN gene product was identified as a weakly acidic 41 kd protein, and the dnaA gene product as a weakly basic 48 kd protein. The synthesis of the dnaN gene product is greatly reduced by insertion of a transposon Tn3 in the dnaA gene and by deletion in the gene at the distal end to the dnaN gene. Temperature-sensitive dnaA mutations, on the other hand, do not affect the synthesis of the dnaN gene product. These results indicate that the synthesis of the dnaN gene product is dependent on the structural integrity of the dnaA gene.Abbreviations kd kilodaltons - SDS sodium dodecyl sulfate     

Expression of chemically synthesized alpha-neo-endorphin gene fused to E. coli alkaline phosphatase.

An alpha-neo-endorphin (alpha NE) gene, which we previously synthesized chemically and inserted into E. coli beta-galactosidase gene of pK013 plasmid, has been excised and fused to E. coli alkaline phosphatase (APase) gene. One of the transformants was named E15/pA alpha NE1. Under the APase gene regulation, APase-alpha NE chimeric protein was expressed at 1.3 X 10(6) molecules per cell, and accounted for about 60% of total cellular proteins. The HPLC pattern of CNBr treated E15/pA alpha NE1 was very simple reflecting the high content of the chimeric protein and low numbers of methionine residues in it. A series of genes encoding APase-alpha NE chimeric proteins in which 30 to 94 C-terminal amino acid residues were replaced by (met)-alpha NE, was cloned in E. coli. Transportation of the chimeric proteins to periplasmic space was studied. All chimeric proteins were apparently processed by signal peptidase but few, if any, was transported to the periplasmic space.     

Novel thick filament protein of chicken pectoralis muscle: the 86 kd protein. II. Distribution and localization

Antibodies specific for the novel 86 kd protein purified from chicken pectoralis myofibrils stained by indirect immunofluorescence the middle third of each half A-band of isolated myofibrils and myotubes. Pectoralis muscle 86 kd protein, like pectoralis C-protein, displayed a fibre-type specific distribution by being restricted to fast twitch fibres and absent in slow tonic and heart muscle fibres. This was demonstrated by immunoblotting experiments with tissue extracts and by immunofluorescence labelling of cryosections. In primary cell cultures prepared from embryonic chicken breast muscle, 86 kd protein, C-protein and myomesin were all detected in post-mitotic myoblasts where fluorescence was found in a cross-striated pattern along strands of nascent myofibrils. Fluorescence due to the 86 kd protein was restricted to myofibrils within myotubes and no significant labelling of the sarcoplasm was evident. Glycerinated fast twitch muscle fibres, after incubation with antibodies to 86 kd protein, revealed in each half of the A-band nine distinctly labelled stripes, spaced about 43 nm apart. Simultaneous incubation of fibres with antibodies against 86 kd protein and C-protein showed a co-localization of the seven C-protein stripes (stripes 5 to 11), with seven stripes of 86 kd protein. The two additional stripes (stripes 3 and 4) labelled by anti-86 kd antibody continued towards the M-band at the same periodicity from the last C-protein stripe (stripe 5). Thus, partial co-localization of two different thick filament proteins is demonstrated and the identity of transverse stripes at positions 3 and 4 attributed in part to the presence of the new 86 kd protein.