Adenovirus E1b-58kd tumor antigen and SV40 large tumor antigen are physically associated with the same 54 kd cellular protein in transformed cells

The adenovirus E1b-58kd tumor antigen has been detected in a physical association with a 54 kilodalton cellular protein in adenovirus-transformed mouse cells. Antibody specific for the E1b-58kd protein coimmunoprecipitates a 54 kd protein from transformed, but not from productively infected, cells. Monoclonal antibody specific for the cellular 54 kd protein coimmunoprecipitates the adenovirus E1b-58kd protein from transformed cell extracts. The same or closely related cellular 54 kd protein, associated with the adenovirus E1b-58kd protein, was present in the SV40 large T antigen-54 kd complex previously detected in SV40-transformed mouse cells. The identity of the 54 kd protein is based on the immunological specificities of the anti-54 kd monoclonal antibodies and partial peptide maps of the 54 kd protein associated with the adenovirus and SV40 tumor antigens. The adenovirus E1b-58kd-54 kd complex, like the SV40 large T antigen-54 kd complex, is heterogeneous in size or mass. While all of the cellular 54 kd protein in the adenovirus-transformed cell extract is found in a complex with the E1b-58kd protein, some of the viral 58 kd antigen is detected in a form not associated with the 54 kd protein. The fact that the adenovirus and Sv40 tumor antigens, both required for transformation, can be found in physical association with the same cellular protein in a transformed cell is a good indication that these two diverse viral proteins share some common mechanisms or functions.     

The E1B 19-kilodalton protein is not essential for transformation of rodent cells in vitro by adenovirus type 5.

The newly constructed adenovirus type 5 mutant in1 carries a single AT base pair insertion immediately after nucleotide position 1715 in the E1B gene sequence which destroys the proximal AUG normally present in E1B messages and prevents production of intact E1B 19-kDa protein in infected cells. We have used in1, variants of in1 containing mutant alleles of viral genes known to enhance transformation frequency, and adenovirus type 5 mutant dl337 (S. Pilder, J. Logan, and T. Shenk, J. Virol. 52:664-671, 1984), in which the sequence between nucleotides 1770 and 1916 within the 19-kDa reading frame is deleted, to test the generally accepted hypothesis that this E1B protein is essential for the transformation of rodent cells and maintenance of the transformed phenotype. We find that these mutants transform rat embryo cells, rat kidney and mouse kidney primary cells, and cells of the 3Y1 rat line with decreased frequencies only when virus is added to these various cells at high input multiplicities of infection. In contrast, when lower doses of virus are used, the mutants transform with wild-type frequencies. Cells infected with higher doses of mutant virus show increased levels of DNA degradation and cell killing compared with those of cells infected with the same levels of wild-type virus, and these effects most likely contribute to the decreased transformation frequencies observed. On the basis of these results and the results of phenotypic analyses of numerous transformants, we propose that the E1B 19-kDa protein is not required for induction and/or maintenance of transformed-cell characteristics in rodent cells infected with adenovirus type 5.     

The 19-kDal protein encoded by early region 1b of adenovirus type 12 is synthesized efficiently in Escherichia coli only as a fused protein

We have constructed recombinant plasmids that direct the synthesis of the Mr 19 000 protein encoded by the adenovirus type 12 (Ad12) E1b region as either a native protein or a protein fused to the amino-terminal portion of the elongation factor EF-TuB in Escherichia coli cells. Using these recombinants, we could synthesize a large amount of the fused protein, while only a small amount of the native Mr 19 000 protein was produced. The failure to synthesize the native Mr 19 000 protein in E. coli cells was ascribed to inefficient translation.     

Tricarboxylate transport in a Cit+ Escherichia coli: evidence for the role of an outer membrane protein

A strain of Escherichia coli of bovine origin able to use tricarboxylates as single carbon source is described. Tricarboxylate utilization (Cit+) and fluorocitrate sensitivity (FCs) could be transferred conjugatively to E. coli K12 and were not plasmid borne. No evidence was found for tct gene products of Salmonella typhimurium. A citrate-inducible outer membrane protein of 21-22 kilodaltons (kd) was found only in Cit+ strains. A protein (21-22kd) protein was also found in wild-type E. coli K12 and in fluorocitrate-resistant mutants of Cit+ strains, but it was present in a cryptic form no longer inducible by citrate. Fluorocitrate-resistant mutants of Cit+ strains were still able to transport citrate by a fluorocitrate-insensitive system. High levels of the 22-kd protein correlated with reduced growth induction times on citrate and with the ability to effectively transport citrate.     

Translation of chloroplast-encoded mRNA: potential initiation and termination signals.

A survey of 196 protein-coding chloroplast DNA sequences demonstrated the preference for AUG and UAA codons for initiation and termination of translation, respectively. As in prokaryotes at every nucleotide position from -25 to +25 (AUG is +1 to +3) and for 25 nucleotides 5' and 3' to the termination codon an A or U is predominant, except for C at +5 and G at +22. A Shine-Dalgarno (SD) sequence (GGAGG or tri- or tetranucleotide variant) was found within 100 bp 5' to the AUG codon in 92% of the genes. In 40% of these cases, the location of the SD sequence was similar to that of the consensus for prokaryotes (-12 to -7 5' to AUG), presumed to be optimal for translation initiation. A SD sequence could not be located in 6% of the chloroplast sequences. We propose that mRNA secondary structures may be required for the relocation of a distal SD sequences to within the optimal region (-12 to -7) for initiation of translation. We further suggest that termination at UGA codons in chloroplast genes may occur by a mechanism, involving 16S rRNA secondary structure, which has been proposed for UGA termination in E. coli.     

Efficient production of a human tumour necrosis factor in Escherichia coli

To examine the effect of altering the nucleotide sequence of the Shine-Dalgarno (SD) region and the spacer sequence between the SD sequence and the AUG translation start signal, several plasmids were constructed which directed the synthesis of mature human tumour necrosis factor (TNF) under the control of the E. coli trp leader promoter. We found that the presence of the SD sequence, AAGGAGGT, which is complementary to the 3' end of 16S rRNA, gave the higher translational efficiency, and also, the presence of the spacer sequence which consists of only A and T residues raised the production of TNF 2-3 fold. The levels of expression of TNF were elevated over 20-fold by the alteration of the SD and spacer sequences and amounted to 20% of the total cellular proteins or approx. 5 X 10(6) molecules of TNF per cell.     

Induction of the synthesis of a 70,000 dalton mammalian heat shock protein by the adenovirus E1A gene product

We have attempted to determine whether any cellular genes are activated as a result of the action of the adenoviral El A gene. The proteins synthesized in uninfected HeLa cells have been compared to those produced in early adenovirus infected cells. At least one protein, absent from uninfected HeLa cells, was synthesized in large amounts following adenovirus infection. This 70 kd protein was not synthesized in cells infected with the E1A mutant d1312, even when the multiplicity of infection with the mutant was such that the only viral gene not expressed was the E1A gene. Thus the induction of the 70 kd protein requires the expression of the viral E1A gene. The 70 kd protein was also induced by heat shock in uninfected cells. The same 70 kd protein is synthesized in 293 cells, a line of human embryonic kidney cells transformed by a fragment of adenovirus DNA. These cells constitutively express the E1A and E1 B genes.     

Detection of Shiga-like toxin on the outer membranes of Shigella species and Escherichia coli K-12

Abstract Outer membranes of Shigella species and E. coli K-12 carrying large invasive plasmids and isogenic non-invasive strains without plasmids were analyzed by SDS-PAGE. The immunoblotting analysis of the outer membrane proteins of these bacteria was performed with monoclonal antibody (mAb) made against A and B subunits of Shiga-like toxin (SLT). The SLT was detected in the outer membranes of S. dysenteriae 1 IDBM11, S. sonnei PNS20, S. flexneri M90T, S. dysenteriae 60R, and E. coli K-12 strain AB2463. The two other E. coli K-12 strains, C600 and 933J were included as controls for low and high toxin producers respectively. The outer membrane protein band of molecular weight 70 kDa was common to all bacterial strains studied. The most prominent band of 70 kDa protein was seen to be present in the high toxin producing plasmidless strain of S. dysenteriae 60R and the lysogenic strain of E. coli 933J. The invasive strains of S. dysenteriae 1 and S. flexneri M90T which carry the large invasive plasmids showed the least prominent band of 70 kDa protein.
The immunoblotting analysis of Shiga-toxin partially purified from the S. dysenteriae 60R strain revealed the absence of 70 kDa band on SDS-PAGE, instead the two dissociated subunits were seen. Furthermore, periplasmic Shiga-toxin proteins also showed the complete dissociation into A and B subunits. However, under the same denaturing conditions, the 70 kDa protein band cross-reacting with mAb against A and B subunits was still present in the outer membranes of all different strains.     

Effect on transformation of mutations in the early region 1b-encoded 21- and 55-kilodalton proteins of adenovirus 5.

It is well established that the adenovirus 5 genes responsible for the initiation and maintenance of the transformed cell reside in the early region 1a and 1b genes, but it remains unclear how the polypeptides encoded in these genes mediate their functions. To probe the function of the early region 1b-encoded 55- and 21-kilodalton (kd) polypeptides during this process, a series of viral mutants was engineered so that they contained deletions or insertions at 5.4, 5.7, 7.9, or 9.6 map units. By means of either an overlap recombination procedure involving H5dl314 (delta 3.7 to 4.6 map units) cleaved with ClaI, or a marker rescue procedure involving H5dl312 (delta 1.2 to 3.8 map units), viral mutants were isolated by their ability to produce plaques on KB cell line 18 cells, which constitutively express only viral early region 1b functions. DNA sequence analysis confirmed that the series of mutants generated differed in their abilities to express the 21- or the 55-kd polypeptides, or both. Upon infection of cloned rat embryo fibroblast cells with viruses containing mutations affecting the 55-kd protein, the transformation frequency decreased as the size of the predicted truncated polypeptide decreased. Although all of the foci generated by the 55-kd protein mutants were indistinguishable from the foci induced by wild-type virus, they displayed an inefficient ability to grow in soft agar, again in relation to the size of the truncated polypeptide. In contrast, if cloned rat embryo fibroblast cells were transfected with viral DNA, the defectiveness in transformation observed after infection with virions was not as dramatic. However, all of the viruses containing 21-kd mutations were transformation defective, regardless of the mode by which the viral nucleic acid was introduced into the cell.     

Identification of early proteins of the human papilloma viruses type 16 (HPV 16) and type 18 (HPV 18) in cervical carcinoma cells.

We have sequenced 1730 bp of human papilloma virus type 18 (HPV 18) DNA containing the open reading frames (ORF) E6, E7, the N-terminal part of E1 and, additionally, 120 bp of the N-terminal part of L1. Based on these sequencing data, together with the human papilloma virus type 16 (HPV 16) DNA sequence published recently, we identified and cloned the ORF E6, E7, E1 and L1 of HPV 18 and the ORF E6, E7, E1, E4, E5, L2 and L1 of HPV 16 into prokaryotic expression vectors. The expression system used provides fusions to the N-terminal part of the MS2 polymerase gene controlled by the heat-inducible lambda PL promoter. Using the purified fusion proteins as immunogens we raised antisera against the proteins encoded by the ORF E6, E7 and E1 of HPV 18 as well as those encoded by the ORF E6, E7, E4 and L1 of HPV 16. By Western blot analysis we could show that the E7 gene product is the most abundant protein in cell lines containing HPV 16 or HPV 18 DNA. It is a cytoplasmic protein of 15 kd in the SiHa and the CaSki cell lines which contain HPV 16 DNA, and 12 kd in the HeLa, the C4-1 and the SW756 cell lines which contain HPV 18 DNA. These results were confirmed by in vitro translation of hybrid-selected HPV 16 and HPV 18 specific poly(A)+ RNA from SiHa, CaSki and HeLa cells. Additionally, these experiments led to the identification of an 11-kd E6 and a 10-kd E4 protein in the CaSki cell line as well as a 70-kd E1 protein in HeLa cells.     

Cloning and characterization of a gene from Rhizobium melilotii 2011 coding for ribosomal protein S1.

A 7 kb chromosomal DNA fragment from R. melilotii was cloned, which complemented temperature-sensitivity of an E. coli amber mutant in rpsA, the gene for ribosomal protein S1 (ES1). From complementation and maxicell analysis a 58 kd protein was identified as the homolog of protein S1 (RS1). DNA sequence analysis of the R. melilotii rpsA gene identified a protein of 568 amino acids, which showed 47% identical amino acid homology to protein S1 from E. coli. The RS1 protein lacked the two Cys residues which had been reported to play an important role for the function of ES1. Two repeats containing Shine-Dalgarno sequences were identified upstream of the structural gene. Binding studies with RNA polymerase from E. coli and Pseudomonas putida located one RNA-polymerase binding site close to the RS1 gene and another one several hundred basepairs upstream. One possible promoter was also identified by DNA sequence comparison with the corresponding E. coli promoter.     

人脑源性神经营养因子基因的克隆及在大肠杆菌中表达

人脑源性神经营养因子基因的克隆及在大肠杆菌中表达何晓龙,路长林,王成海（第二军医大学神经生物学教研室,上海２００４３３）关键词神经营养因子;基因克隆脑源性神经营养因子（ｂｒａｉｎ－ｄｅｒｉｖｅｄｎｅｕｒｏｔｉｃ…ｉ。血。ｔｏｒ,ＢＤ贾助是Ｂａｄｅ等人...     

The effect of Escherichia coli ribosomal protein S1 on the translational specificity of bacterial ribosomes

Ribosomes from Gram-negative bacteria such as Escherichia coli exhibit non-specific translation of bacterial mRNAs. That is, they are able to translate mRNAs from a variety of sources in a manner independent of the "strength" of the Shine-Dalgarno region, in contrast to ribosomes from many Gram-positive bacteria, such as Bacillus subtilis, which show specific translation in only being able to translate other Gram-positive mRNA, or mRNAs that have "strong" Shine-Dalgarno regions. There is an evolutionary correlation between the translational specificity and the absence of a protein analogous to E. coli ribosomal protein S1. The specificity observed with B. subtilis ribosomes is a function of their 30 S subunit which lacks S1; translation of Gram-negative mRNA can occur with heterologous ribosomes containing the 30 S subunit of E. coli ribosomes and the 50 S subunit of B. subtilis ribosomes. However, the addition of E. coli S1 alone to B. subtilis ribosome does not overcome their characteristic inability to translate mRNA from Gram-negative organisms. By contrast, the removal of S1 from E. coli ribosomes results in translational behavior similar to that shown by B. subtilis ribosomes in that the S1-depleted E. coli ribosomes can translate mRNA from Gram-positive sources in the absence of added S1, although addition of S1 stimulates further translation of such mRNAs by the E. coli ribosomes.     

The tonB gene of Serratia marcescens: sequence, activity and partial complementation of Escherichia coli tonB mutants

The TonB protein plays a key role in the energy-coupled transport of iron siderophores, of vitamin B12, and of colicins of the B-group across the outer membrane of Escherichia coli. In order to obtain more data about which of its particular amino acid sequences are necessary for TonB function, we have cloned and sequenced the tonB gene of Serratia marcescens. The nucleotide sequence predicts an amino acid sequence of 247 residues (Mr 27,389), which is unusually proline-rich and contains the tandem sequences (Glu-Pro)5 and (Lys-Pro)5. In contrast to the TonB proteins of E. coli and Salmonella typhimurium, translation of the S. marcescens TonB protein starts at the first methionine residue of the open reading frame, which is the only amino acid removed during TonB maturation and export. Only the N-terminal sequence is hydrophobic, suggesting its involvement in anchoring the TonB protein to the cytoplasmic membrane. The S. marcescens tonB gene complemented an E. coli tonB mutant with regard to uptake of iron siderophores, and sensitivity to phages T1 and phi 80, and to colicins B and M. However, an E. coli tonB mutant transformed with the S. marcescens tonB gene remained resistant to colicins Ia and Ib, to colicin B derivatives carrying the amino acid replacements Val/Ala and Val/Gly at position 20 in the TonB box, and they exhibited a tenfold lower activity with colicin D. In addition, the S. marcescens TonB protein did not restore T1 sensitivity of an E. coli exbB tolQ double mutant, as has been found for the overexpressed E. coli TonB protein, indicating a lower activity of the S. marcescens TonB protein. Although the S. marcescens TonB protein was less prone to proteolytic degradation, it was stabilized in E. coli by the ExbBD proteins. In E. coli, TonB activity of S. marcescens depended either on the ExbBD or the TolQR activities.