Daniel Nathans

DNA binding activities of three murine Jun proteins: Stimulation by Fos

Three members of the Jun/AP-1 family have been identified in mouse cDNA libraries: c-Jun, Jun-B, and Jun-D. We have compared the DNA binding properties of the Jun proteins by using in vitro translation products in gel retardation assays. Each protein was able to bind to the consensus AP-1 site (TGACTCA) and, with lower affinity, to related sequences, including the cyclic AMP response element TGACGTCA. The relative binding to the oligonucleotides tested was similar for the different proteins. The Jun proteins formed homodimers and heterodimers with other members of the family, and they were bound to the AP-1 site as dimers. When Fos translation product was present, DNA binding by Jun increased markedly, and the DNA complex contained Fos. The C-terminal homology region of Jun was sufficient for DNA binding, dimer formation, and interaction with Fos. Our general conclusion is that c-Jun, Jun-B, and Jun-D are similar in their DNA binding properties and in their interaction with Fos. If there are functional differences between them, they are likely to involve other activities of the Jun proteins.

Identification of a set of genes expressed during the G0/G1 transition of cultured mouse cells.

To identify previously undetected genes that may be involved in the transition from a resting state (G0) to a proliferative state (G1) of mammalian cells, we set out to isolate cDNA clones derived from mRNAs that appear in serum‐stimulated cells in the absence of protein synthesis. A lambda cDNA library was prepared using poly(A)+ RNA from BALB/c 3T3 cells that had been brought to quiescence and subsequently stimulated with serum in the presence of cycloheximide. Approximately 50 000 recombinant phage plaques were screened, and 357 clones were isolated that hybridized to probes derived from stimulated‐cell RNA but not to probes from resting‐cell RNA. Cross hybridization analysis showed that four RNA sequence families account for approximately 90% of these clones. One of the clones hybridized to an actin probe; none hybridized to any of 13 oncogene probes tested. Five different RNAs that appear to be previously uncharacterized have been further analyzed. These RNAs accumulate and decay rapidly following stimulation by serum or purified growth factors, or by a tumor promoter, and they are superinduced by serum in the presence of cycloheximide. Three of the RNAs could be enriched by hybridization to cDNAs and translated in vitro, yielding proteins of approximately 43, 40 and 35 kd, respectively.

A naturally occurring truncated form of FosB that inhibits Fos/Jun transcriptional activity

Fos and Jun transcription factors are induced by a variety of extracellular signaling agents. We describe here an unusual member of the Fos family that is also induced, namely, a truncated form of FosB (delta FosB) missing the C-terminal 101 amino acids of FosB. delta FosB retains the dimerization and DNA-binding activities of FosB but has lost the ability in transfection assays to activate a promoter with an AP-1 site and to repress the c-fos promoter. Rather, delta FosB inhibits gene activation by Jun or Jun + Fos and inhibits repression of the c-fos promoter by FosB or c-Fos, presumably by competing with full-length Fos proteins at the steps of dimerization with Jun and binding to DNA. In stimulated cells delta FosB may act to limit the transcriptional effects of Fos and Jun proteins.

A Suggested nomenclature for bacterial host modification and restriction systems and their enzymes

Abstract In the proposed nomenclature restriction-modification systems are named according to host organism and strain. Different R-M systems in a single host are designated by Roman numerals. Restriction nucleases and modification methylases are given the general names endonuclease R and methylase M, followed by their R-M system name.

Studies of Simian virus 40 DNA. VII. A cleavage map of the SV40 genome

A physical map of the Simian virus 40 genome has been constructed on the basis of specific cleavage of Simian virus 40 DNA by bacterial restriction endonucleases. The 11 fragments produced by enzyme from Hemophilus influenzae have been ordered by analysis of partial digest products and by analysis of an overlapping set of fragments produced by enzyme from Hemophilus parainfluenzae. In addition, the single site in SV40 DNA cleaved by the Escherichia coli RI restriction endonuclease has been located. With this site as a reference point, the H. influenzae cleavage sites and the H. parainfluenzae cleavage sites have been localized on the map.

Inhibition of aminoacyl-sRNA binding to ribosomes by tetracycline☆

Abstract Tetracycline blocked the synthesis of polyphenylalanine from phenylalanyl-sRNA. This inhibition was reversed by increased ribosomes plus poly U in the incubation mixture but not by increased aminoacyl-sRNA or supernatant fraction. In contrast to the lack of effect of puromycin and chloramphenicol, tetracycline reduced the binding of N-acetyl-phenylalanyl-sRNA to ribosomes, whereas the binding of poly U to ribosomes was unaffected. Although polyphenylalanine synthesis was inhibited almost completely by tetracycline, aminoacyl-sRNA binding to ribosomes was inhibited maximally about 50 per cent. We suggest that tetracycline may combine with ribosomes in such a way as to obstruct one of the two sites for the binding of aminoacyl-sRNA.

Functional differences between Stat3alpha and Stat3beta.

Stat3beta is a short form of Stat3 that differs from the longer form (Stat3alpha) by the replacement of the C-terminal 55 amino acid residues of Stat3alpha by 7 residues specific to Stat3beta. In COS cells transfected with Stat3 expression plasmids, both Stat3alpha and Stat3beta were activated for DNA binding and transcription by the same set of growth factors and cytokines and both, when activated, formed homodimers and heterodimers with Stat1. Only Stat3beta was active in the absence of added cytokine or growth factor. Activation of each form, including constitutive activation of Stat3beta, was correlated with the phosphorylation of tyrosine 705. Activated Stat3beta in transfected COS cells was more stable and had greater DNA-binding activity than activated Stat3alpha. However, relative to DNA-binding activity, Stat3alpha showed greater transcriptional activity than Stat3beta. A mutant of Stat3alpha lacking its highly acidic C-terminal 48 amino acids had properties indistinguishable from Stat3beta. We conclude that Stat3alpha and Stat3beta have significantly different properties due to the presence or absence of the acidic C-terminal tail of Stat3alpha rather than the C-terminal sequence peculiar to Stat3beta. In addition to its effect on transcription, we speculate that the acidic tail may destabilize the active dimeric form of Stat3alpha, resulting in lower DNA-binding activity of the Y705-phosphorylated form compared to Stat3beta and in more rapid dephosphorylation.

Mutational analysis of simian virus 40 T antigen: isolation and characterization of mutants with deletions in the T-antigen gene.

A series of mutants of simian virus 40 has been constructed with deletions in the coding sequence for large T antigen. Nucleotide sequence analysis indicates that 4 mutants have in-phase and 11 have out-of-phase deletions. Mutant DNAs were assayed for the following activities: the ability to form plaques, the ability to produce T antigen as scored by indirect immunofluorescence, viral DNA replication, and morphological transformation of rat cells. Two viable mutants were found, and these had deletions confined to the carboxyl terminus of T antigen. Only those mutants coding for polypeptides greater than 40% of the length of wildtype T antigen produced detectable nuclear fluorescence. The two viable mutants with deletions in the carboxyl terminus of the protein retained the ability both to replicate their DNA, although at a reduced level, and to transform nonpermissive cells. Mutants with sequence changes that result in the loss of more than 117 amino acids from the carboxyl terminus were not viable and were also defective in the DNA replication and transformation functions of T antigen, although several produced detectable nuclear fluorescence. These functions were also sensitive to the removal of amino acids near the amino terminus and in the middle of the protein.

Deletion mutants of simian virus 40 generated by enzymatic excision of DNA segments from the viral genome

Abstract Deleted genomes of simian virus 40 have been constructed by enzymatic excision of specific segments of DNA from the genome of wild-type SV40 † . For this purpose, a restriction endonuclease from Hemophilus influenzae (endo R · Hind III) was used. This enzyme cleaves SV40 DNA into six fragments, which have cohesive termini. Partial digest products were separated by electrophoresis in agarose gel and subsequently cloned by plaque formation in the presence of complementing temperature-sensitive mutants of SV40. Individual deletion mutants generated in this way were mapped by analysis of DNA fragments produced by endo R · Hin d digestion of their deleted genomes, and by heteroduplex mapping. Two types of deletions were found: (1) “excisional” deletions, in which the limits of the deleted segment corresponded to Hin dIII cleavage sites, and (2) “extended” deletions, in which the deleted segment extended beyond Hin dIII cleavage sites. Excisionally deleted genomes presumably arose by cyclization of a linear fragment via cohesive termini generated by endo R · Hin dIII whereas genomes with extended deletions probably were generated by intramolecular recombination near the ends of linear fragments. Of the nine mutants analyzed, two had deletions in the “early” region of the SV40 genome, six had deletions in the “late” region, and one had a deletion that spanned both regions.

Mapping temperature-sensitive mutants of simian virus 40: Rescue of mutants by fragments of viral DNA☆

Abstract Five temperature-sensitive (ts) mutants of simian virus 40 (SV40), isolated and characterized by Tegtmeyer and Ozer (1971) , have been mapped by marker rescue using endo R fragments of wild-type SV40 DNA. For each mutant a specific fragment corrected the ts defect, from which we infer that the mutation is within the genome segment corresponding to the active fragment. Since the position of each fragment in the SV40 cleavage map is known, the mutational sites could be localized. Of the five ts mutants examined, two were “early” mutants and were in complementation group A of Tegtmeyer; these mapped in contiguous Hin fragments H (tsA30) and I (tsA28), which had been shown previously to be part of the “early” region of the SV40 genome. Three ts mutants were “late” mutants and were in complementation group B of Tegtmeyer; these mapped in contiguous Hin fragments F (tsB8), J (tsB4), and G (tsB11), which had been shown previously to be part of the “late” region of the SV40 genome.