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Biomedical subjects

D Nathans

Publications and source records attributed to D Nathans.

At least 55 records · Page 3Linked to original sources
PubMed1985

Nucleotide sequence of murine ornithine decarboxylase mRNA.

Ornithine decarboxylase (OrnDCase; L-ornithine carboxy-lyase, EC 4.1.1.17) is the first and rate-limiting enzyme in the biosynthesis of polyamines in mammalian cells. During cell growth the enzyme is regulated by rapid changes in the level of its mRNA and protein. To explore the molecular basis of these changes, we cloned a full-length cDNA copy of the major 2.4-kilobase OrnDCase mRNA from mouse cells and determined its sequence. The cDNA contains 2465 nucleotides derived from OrnDCase mRNA, consisting of a 737-nucleotide-long 5' noncoding segment, a coding segment of 1383 nucleotides terminated by a TAG triplet, and a 342-nucleotide 3' noncoding segment. The encoded protein of 461-amino acid residues has a molecular weight of 51,105 and has a potential site for phosphorylation by casein kinase II. In the unusually long 5' leader sequence, there are four ATG triplets, each of which is followed by an in-phase termination signal; the presence of these upstream ATGs could explain the low in vitro translational activity of OrnDCase mRNA reported earlier. A restriction digest of mouse genomic DNA was probed with a defined OrnDCase coding sequence, revealing a multimembered family of OrnDCase-related genes.

Amino Acid Sequence↗
PubMed1985

Purification of simian virus 40 large T antigen by immunoaffinity chromatography.

Simian virus 40 large T antigen from lytically infected cells has been purified to near homogeneity by immunochromatography of the cell extract on a protein A-Sepharose-monoclonal antibody column. The resulting T antigen retains biochemical activity; i.e., it hydrolyzes ATP and binds to simian virus 40 DNA at the origin of replication.

Animals↗
PubMed1984

Isolation of cloned cDNA encoding mammalian ornithine decarboxylase.

During the growth of mammalian cells the level of ornithine decarboxylase ( OrnDCase ; L-ornithine carboxy-lyase, EC 4.1.1.17), the first enzyme in polyamine biosynthesis, undergoes rapid changes. As an initial step in the study of possible genetic mechanisms involved in these changes, we have isolated cDNA clones encoding OrnDCase . To obtain RNA enriched for OrnDCase messenger, mouse myeloma cells that overproduce OrnDCase were selected in the presence of the OrnDCase inhibitor, difluoromethylornithine. A pBR322 cDNA library was prepared from poly(A)+ RNA isolated from difluoromethylornithine-resistant cells, and the library was probed with [32P]cDNA representing mRNA sequences from resistant or parental (sensitive) cells. All clones hybridizing preferentially to the resistant cell probe shared nucleotide sequences. A representative clone containing 1.1 kilobases of cDNA was shown to encode OrnDCase sequences by in vitro translation of hybrid-selected mRNA followed by precipitation of the translation products with anti- OrnDCase antiserum. Using this cDNA clone as a probe, we found that mouse DNA yielded several restriction fragments that react with the OrnDCase cDNA. In the difluoromethylornithine-resistant myeloma cells, one of these DNA segments is amplified and the level of OrnDCase mRNA is greatly increased compared with that in parental plasmacytoma cells. The level of OrnDCase mRNA is also increased in cultured 3T3 cells stimulated with serum and in mouse kidneys after administration of androgen, indicating that OrnDCase gene transcription and/or mRNA stability are regulated during cell growth.

Animals↗
PubMed1984

Nucleotide sequence of a growth-related mRNA encoding a member of the prolactin-growth hormone family.

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."

Amino Acid Sequence↗
PubMed1984

Simian virus 40 mutant T antigens with relaxed specificity for the nucleotide sequence at the viral DNA origin of replication.

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.

Animals↗
PubMed1983

Growth-related changes in specific mRNAs of cultured mouse cells.

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.

Animals↗
PubMed1983

Suppression of a VP1 mutant of simian virus 40 by missense mutations in serine codons of the viral agnogene.

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.

Codon↗
PubMed1983

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.

Amino Acid Sequence↗
PubMed1983

Biochemical activities of T-antigen proteins encoded by simian virus 40 A gene deletion mutants.

We have analyzed T antigens produced by a set of simian virus 40 (SV40) A gene deletion mutants for ATPase activity and for binding to the SV40 origin of DNA replication. Virus stocks of nonviable SV40 A gene deletion mutants were established in SV40-transformed monkey COS cells. Mutant T antigens were produced in mutant virus-infected CV1 cells. The structures of the mutant T antigens were characterized by immunoprecipitation with monoclonal antibodies directed against distinct regions of the T-antigen molecule. T antigens in crude extracts prepared from cells infected with 10 different mutants were immobilized on polyacrylamide beads with monoclonal antibodies, quantified by Coomassie blue staining, and then assayed directly for T antigen-specific ATPase activity and for binding to the SV40 origin of DNA replication. Our results indicate that the T antigen coding sequences required for origin binding map between 0.54 and 0.35 map units on the SV40 genome. In contrast, sequences closer to the C terminus of T antigen (between 0.24 and 0.20 map units) are required for ATPase activity. The presence of the ATPase activity correlated closely with the ability of the mutant viruses to replicate and to transform nonpermissive cells. The origin binding activity was retained, however, by three mutants that lacked these two functions, indicating that this activity is not sufficient to support either cellular transformation or viral replication. Neither the ATPase activity nor the origin binding activity correlated with the ability of the mutant DNA to activate silent rRNA genes or host cell DNA synthesis.

Adenosine Triphosphatases↗
PubMed1982

Local mutagenesis within deletion loops of DNA heteroduplexes.

An efficient method has been developed to generate base substitution mutations within deletion loops of DNA heteroduplexes. This method utilizes a heteroduplex formed between a deletion mutant cloned in a plasmid vector and its wild-type counterpart from which two restriction sites had been removed from the vector. The heteroduplex is exposed to sodium bisulfite to deaminate cytosine residues in the single-stranded loop, and the mutagenized plasmid DNA is used to transform a strain of bacteria lacking the enzyme uracil N-glycosylase. Pooled progeny DNA is digested with the two restriction enzymes, whose sites had been mutated in the wild-type plasmid, to eliminate the original deletion mutant DNA. Point mutants with C . G-to-T . A transitions are obtained at high frequency after a second transformation. To test the feasibility of the approach, the tetracycline resistance gene of pBR322 was chosen as the target sequence. It was found that the proportion of tetracycline-sensitive transformants increased as both the size of the heteroduplex loop and the time of incubation with the mutagen increased and this varied from 20% up to 70%. Nucleotide sequence analysis of several tetracycline-sensitive mutants confirmed that C-to-T transitions had been produced in the segment of DNA corresponding to the deletion loop.

Base Sequence↗
PubMed1980

Isolation of mutants of an animal virus in bacteria.

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.

Animals↗