An mRNA encoding a helix-loop-helix pro- tein that we have named HLH462 is induced in mouse 3T3 cells as part of the immediate early transcriptional response to growth factors and other signaling agents. The RNA is present in a number of mouse tissues and in the developing mouse fetus. The HLH462 gene has been mapped by interspecific backcross analysis to the distal region of mouse chromosome 4. In its helix-loop-helix region HLH462 is closely related to the Id protein and the Drosophila emc protein. Like Id, HLH462 lacks a basic region required for DNA binding, and it inhibits the DNA-binding activities of other helix-loop-helix proteins. On the basis of its structural and functional similarity to Id, we suggest that HLH462 may inhibit the activities of helix-loop- helix transcription factors during the cellular growth response and during development.

Reviews in Medical VirologyVolume 9, Issue 2 p. 75-81 Classic Paper Specific cleavage of simian virus 40 DNA by restriction endonuclease of Hemophilus influenzae†‡ R. J. Roberts, Corresponding Author R. J. Roberts Reviewer New England Biolabs, 32 Tozer Road, Beverly, MA 01915, USADirector of Research, New England Biolabs, 32 Tozer Road, Beverly, MA 01915, USA.Search for more papers by this author R. J. Roberts, Corresponding Author R. J. Roberts Reviewer New England Biolabs, 32 Tozer Road, Beverly, MA 01915, USADirector of Research, New England Biolabs, 32 Tozer Road, Beverly, MA 01915, USA.Search for more papers by this author First published: 26 May 1999 https://doi.org/10.1002/(SICI)1099-1654(199904/06)9:2<75::AID-RMV250>3.0.CO;2-BCitations: 1 † Reproduced from Proc. Natl Acad. Sci. USA68, 2913-2917 (1971) with kind permission of Daniel Nathans. ‡ Kathleen Danna and David Nathans AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume9, Issue2April/June 1999Pages 75-81 RelatedInformation

Science is to be commended for dedicating a portion of its 23 June issue to the topic "Conduct in Science" and for including in it Gary Taubes's descriptions of novel approaches to formal teaching about research conduct. I wish to make two comments, one general, the other partly self-serving (not untypical for a scientist). The special section emphasizes the biomedical sciences, where NIH training grants predominate, with their concomitant requirement for some formal lecture-teaching exposure to ethical issues. There is, however, no coverage of the physical sciences, where training grants are rare. As the only chemist member of the original Institute of Medicine-National Academy of Sciences committee on the ethical conduct of research, I called attention to this operational difference in research support, which led me to suggest that recipients of research grants should also be obligatorily exposed to such instruction. This has not yet happened, which makes the question of pedagogic experimentation even more relevant. Taubes's description of pedagogic experiments omits one that I believe merits some emphasis, as it also serves to enlighten the general lay public about conduct in scientific research. The general public knows little enough of what we do, but it knows even less how we do it. I am currently working on a tetralogy of novels in the infrequently used literary genre of "science-in-fiction" (not science fiction) to illustrate in an accurate way in the guise of fiction the behavior of contemporary research scientists. The reception of the first novel, Cantor's Dilemma (Penguin, New York, 1991) has convinced me that "science-in-fiction" is a pedagogic tool well worth implementing, as it can cover the gamut from the general public to graduate students and postdocs. In the afterword of Cantor's Dilemma, I said, "Publications, priorities, the order of the authors, the choice of the journal, the collegiality and the brutal competition, academic tenure, grantsmanship, the Nobel Prize, Schadenfreude-these are the soul and baggage of contemporary science. To illustrate them ... I write about behavior and attitudes surely more common than we like to admit." This novel has been translated into six languages, and was serialized daily in Germany's largest newspaper, the Frankfurter Allgemeine Zeitung. More relevant to the coverage of teaching such issues, it has become a text or recommended reading in many American colleges and universities. Finally, nowhere in the otherwise extremely well-done coverage of scientific conduct do I find comment on the gender aspects of our science research culture, other than tangentially through description of the legal travails surrounding Sarvamangala Devi. There is more to it than just featuring women as whistleblowers or plaintiffs. How to compete on the tenure-track treadmill while pregnant and how the rest of the scientific establishment responds are issues well worth exploring as part of a broad overview of conduct in science, rather than in a group of articles about the special problems facing women in a tough laboratory science. I have made this a key element in my science-in-fiction series and have found from my lectures and even book reviews that it raises more questions and comments than any other. Cart Djerassi Department of Chemistry, Stanford University, Stanford, CA 94305-5080, USA

A growth factor that stimulates the proliferation of endothelial cells from human umbilical vein but is not mitogenic for fibroblastic cells is present in medium conditioned by the mouse neuroblastoma cell line NB41. In a partially purified preparation, factor activity coeluted from a reverse-phase high-pressure liquid chromatography (HPLC) column with a reduced protein of about 24 kd. Activity recovered following electrophoresis of HPLC fractions corresponded to protein of 43-51 kd in the absence of reducing agent and to protein of 23-29 kd after reduction. Antiserum raised against a peptide corresponding to the putative N-terminal amino acid sequence of the 24-kd protein reacted with the 24-kd protein and with a protein of about 47 kd in the nonreduced preparation. After N-glycanase treatment, the immunoreactive 24-kd protein had a mobility corresponding to 19 kd. We infer that the native NB41 factor is a glycosylated dimer whose biochemical and biological properties distinguish it from other endothelial cell growth factors.

Proliferin (PLF) is a PRL-related glycoprotein secreted by a number of mouse cell lines and by minced mouse placenta. To further investigate the hormone-like characteristics of PLF, we have determined the site of PLF synthesis and storage in the placenta and its concentration in blood serum during pregnancy. By immunohistochemical staining and in situ hybridization PLF protein and messenger RNA (mRNA) were localized to the trophoblastic giant cells. Individual cells contained both PLF and placental lactogen II. Trophoblastic giant cells appear to secrete PLF into the circulation since PLF was found at levels up to 5 micrograms/ml in the serum of pregnant mice by RIA and at somewhat lower concentrations in the amniotic fluid. Moreover, the serum concentration of PLF during pregnancy varied directly with the level of PLF mRNA in the placenta and with the number of placentas per animal. These findings are consistent with the hypothesis that PLF is a placental hormone; its function is not known.

We constructed a collection of linker insertion mutants in the simian virus 40 (SV40) genome and studied several of these with changes limited to a part of the large T antigen gene corresponding to an amino acid sequence shared with other ATPases. Two of these mutants were found to have a novel phenotype in that they could not be complemented for plaque formation by a late-region deletion mutant. These two mutants, in contrast to other mutants in this region, were able to transform rat cells in culture at a frequency close to that of the wild-type gene. The noncomplementing mutants were found to be potent inhibitors of SV40 DNA replication despite the presence of wild-type T antigen in the transfected cells. This inhibition was shown to be the result of the introduced mutations in the large T antigen gene. We conclude that the large T antigens of the noncomplementing mutants can act as inhibitors of SV40 DNA replication.

Proliferin is a recently described, prolactin-related protein whose mRNA appears in several murine cell lines during active growth. We have surveyed a number of mouse organs or tissues for the presence of mRNAs that hybridize to cloned proliferin cDNA. Of the tissues tested, only the placenta yielded proliferin-related mRNA. This placental RNA is about 1 kilobase in length, increases sharply between days 8 and 10 of pregnancy, and then gradually declines through day 18. It is more abundant in RNA extracted from the fetal, compared to the maternal, part of the placenta. From a cDNA plasmid library prepared from poly(A)+ placental RNA, two types of proliferin-related clones were isolated, differing in intensity of hybridization to proliferin cDNA. By nucleotide sequence analysis, a strongly hybridizing clone was found to be nearly identical to the proliferin cDNA clone isolated from a library prepared from mRNA of a growing mouse fibroblastic cell line. Using an antiserum prepared against a synthetic proliferin fusion protein, we show that proliferin is secreted as a glycoprotein by minced placental tissue and that it differs from mouse placental lactogen. We conclude that proliferin is a placental hormone that is synthesized in certain mouse cell lines during active growth. Its function during pregnancy and during the growth of cultured cells is presently unknown.

Base substitution of the ori region of simian virus 40 leads to plaque morphology mutants with markedly decreased DNA replication. Second-site mutations within the simian virus 40 T antigen gene suppress the plaque phenotype and replication defect of base-substituted ori mutants. Two second-site mutations have been mapped to a small segment of the T antigen gene, just beyond the distal splice junction. DNA sequence analysis revealed a single missense change in this segment of the T antigen gene of each of these second-site revertants, leading to a change in codon 157 in one case and codon 166 in the other. The mutant T antigens displayed relaxed specificity for the ori signal, i.e., they can function with several variously modified ori sequences, including those with small nucleotide deletions or insertions that are inactive for replication when coupled with wild-type T antigen. Thus a region of T antigen has been identified that appears to be intimately involved in vivo in binding to the ori sequence to initiate viral DNA replication.

As part of the proliferative response to serum, mouse 3T3 cells produce a set of growth-related mRNAs identified by hybridization to cloned cDNAs. One of these mRNAs, which is about 1 kilobase long, appears within a few hours after stimulation of resting cells with serum or platelet-derived growth factor and reaches a high level during the transition from the G1 to the S phase of growth. This mRNA is translated in vitro into a protein of approximately 25 kilodaltons. The corresponding cloned cDNA of 791 base pairs has been sequenced; it contains a single open reading frame that encodes a protein of 224 amino acids with extensive sequence homology to mammalian prolactins. The initial 29-amino acid segment of the encoded protein resembles the signal sequences of prehormones. That the growth-related protein is not mouse prolactin is indicated by comparison of its predicted amino acid composition with that of mouse prolactin and by the distinct fragment patterns seen when restricted mouse DNA is probed with the cloned cDNA or rat prolactin cDNA. Therefore, the growth-related protein appears to be a new member of the prolactin-growth hormone family. Because of its relationship to prolactin and growth hormone and its association with cell proliferation, the protein has been called "proliferin."

We isolated second-site revertants of a partially defective VP1 mutant of simian virus 40. The suppressing mutation in each of these pseudorevertants was mapped to the viral agnogene. Of six independently isolated pseudorevertants, all had a missense mutation in a serine codon, near the beginning of the agnogene, that would cause replacement of serine at position 7, 11, or 17 in the agnoprotein by a hydrophobic amino acid. Our results suggest that the agnoprotein interacts in a specific way with VP1 during the late stages of viral development.

A cDNA plasmid library has been constructed from the poly(A)+ RNA present in BALB/c3T3 cells after serum stimulation. Of 3,500 clones tested, approximately 0.5% contained inserts corresponding to mRNAs present at higher levels in serum-stimulated BALB/c 3T3 cell cultures than in quiescent cultures. Most of these RNA species increased 2- to 5-fold, and the kinetics of increase for various RNAs differed. One clone (28H6) hybridized to a 1-kilobase RNA species that is present at barely detectable levels in resting cells but is increased at least 15- to 20-fold after serum stimulation, reaching a maximal level coincident with the onset of DNA synthesis. This RNA was at a high level in proliferating cells but decreased rapidly as cells reached confluence. 28H6 RNA was also increased in resting cells infected with simian virus 40 or stimulated with platelet-derived growth factor.

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.

We have characterized a series of simian virus 40 (SV40) mutants with defined deletions or base-pair substitutions in the viral regulatory region near the origin of DNA replication. This segment of DNA is thought to contain three binding sites of differing affinity for the SV40 early protein, large T antigen, which is involved in initiation of viral DNA replication and autoregulation of early gene expression. Cells infected with viruses containing mutations in the high affinity binding site I were found to have a moderate, generally cold-sensitive defect in viral DNA replication and late protein synthesis that correlates with the temperature-dependence of their plaque size. In addition, the mutants overproduce early RNA and T antigen at various temperatures, suggesting a defect in autoregulation of early gene transcription. These changes appear to be related to T antigen functions at the binding site, since a second-site mutation in the T antigen gene restores viral DNA replication and gene expression to normal. Moreover, regulatory segments from mutant DNAs show decreased in vitro binding of T antigen to the high affinity site I. We conclude that T antigen binding to site I is involved in autoregulation of early gene transcription but is not essential for viral DNA replication. These properties contrast with previously reported properties of site II mutants, some of which show marked defects in viral DNA replication.

METHODS THAT GENERATE BASE SUBSTITUTIONS ...................................... Fragment Mutagenesis followed by Recombination .................................................. Site Mutagenesis of Intact Genomes ....................................... Incorporation of nucleotide analogues ............................. Bisulfte reaction at single-stranded gaps .................................................................... Nucleotide misincorpration ........................................... Mutant Construction with Synthetic Oligonucleotides ... ..................................................

Mutants of animal viruses can be isolated in bacteria by recombinant DNA methods. Since no viral functions are required for propagation of recombinants in bacteria, viral mutants with lethal changes in cis- or trans-acting elements can be isolated, as well as partially or conditionally defective mutants. In the cases of viruses with small DNA genomes, such as the tumorigenic simian virus 40 (SV40), the entire viral DNA can be inserted into the bacterial plasmid pBR322 and cloned in Escherichia coli. Recombinant plasmids with a single copy of SV40 DNA cause morphological transformation of mouse cells in culture with the same efficiency as SV40 DNA isolated from virus-infected monkey cells, but the recombinant DNA is noninfectious and replicates poorly in permissive cells. However, SV40 DNA excised from the plasmid replicates as well as authentic viral DNA and is fully infectious. SV40 mutants with small deletions or base substitutions have been isolated by in vitro site-specific or random local mutagenesis of recombinant DNA followed by cloning in E. coli. Many of the mutants thus isolated are defective in specific viral functions.