Probable cloning artefacts previously interpreted as unusual leader sequences of rodent ornithine decarboxylase mRNAs--a cautionary tale

Messenger RNAs that have structurally unusual 5' leaders attract interest and provoke conjecture. Cloning and sequencing of two rodent ornithine decarboxylase (ODC) cDNAs, those for mouse [Kahana and Nathans, Proc. Natl. Acad. Sci. USA 82 (1985) 1673-1677] and, recently as published in this journal, for rat [Van Kranen et al., Gene 60 (1987) 145-155], have indicated the presence of such features. In both cases, the leader is unusually long and contains multiple AUG start codons preceding that which encodes the N terminus of the protein. In addition, the leader of the rat clone contains a 54-nt perfect inverted repeat. Because ODC expression appears to be regulated translationally, functional implications immediately suggest themselves. Certain unusual features of the mouse cDNA have proven artefactual [Brabant et al., Proc. Natl. Acad. Sci. USA 85 (1988) 2200-2204; Katz and Kahana, J. Biol. Chem. 263 (1988) 7604-7609]. It is likely that the putative leader sequence of rat ODC cDNA also resulted from a cloning artefact.     

Amino-acid sequence of the cytochrome-b5-like heme-binding domain from Hansenula anomala flavocytochrome b2

Flavocytochrome b2 (L-lactate dehydrogenase) from baker's yeast is composed of two structural and functional domains. Its first 100 residues constitute the heme-binding core, which is homologous to cytochrome b5 [B. Guiard, O. Groudinsky & F. Lederer (1974) Proc. Natl Acad. Sci. USA 71, 2539-2543]. We report here the amino acid sequence of the heme-binding domain isolated by tryptic proteolysis of Hansenula anomala flavocytochrome b2. The sequence was established by automated degradation of the whole fragment and of peptides obtained by CNBr cleavage at the unique tryptophan and by proteolysis with thermolysin and endoproteinase Lys C. As isolated, the domain consists of 84 residues without any sulfur amino acids. It shows 49 identities with the heme-binding domain from Saccharomyces cerevisiae and 28 with beef microsomal cytochrome b5. Using the recently published three-dimensional structure of S. cerevisiae flavocytochrome b2 [Z-x. Xia, N. Shamala, P. H. Bethge, L. W. Lim, H. D. Bellamy, N. H. Xuong, F. Lederer and F. S. Mathews (1987) Proc. Natl Acad. Sci. USA 84, 2629-2633], it can be seen that there are only positively charged side chains close to the accessible heme edge, the only negative charges in that area being those of the heme propionates. The implications of this result are discussed in the light of Salemme's model for the cytochrome b5/cytochrome c complex [F. R. Salemme (1976) J. Mol. Biol. 102, 563-568].     

A natural oocyte component required for the reprogramming of somatic cell nuclei

Comment on: Jullien J, et al. Proc Natl Acad Sci USA 2010; 107:5483-8.     

Consistencies of individual DNA base-amino acid interactions in structures and sequences.

Amino acid-amino acid interaction energies have been derived from crystal structure data for a number of years. Here is reported the first derivation of normalized relative interaction from binding data for each of the four bases interacting with a specific amino acid, utilizing data from combinatorial multiplex DNA binding of zinc finger domains [Desjarlais, J. R. and Berg, J. M. (1994) Proc. Natl. Acad. Sci. USA, 91, 11099-11103]. The five strongest interactions are observed for lysine-guanine, lysine-thymine, arginine-guanine, aspartic acid-cytosine and asparagine-adenine. These rankings for interactions with the four bases appear to be related to base-amino acid partial charges. Also, similar normalized relative interaction energies are derived by using DNA binding data for Cro and lambda repressors and the R2R3 c-Myb protein domain [Takeda, Y., Sarai, A. and Rivera, V. M. (1989) Proc. Natl. Acad. Sci. USA, 86, 439-443; Sarai, A. and Takeda, Y. (1989) Proc. Natl. Acad. Sci. USA, 86, 6513-6517; Ogata, K. et al. (1995) submitted]. These energies correlate well with the combinatorial multiplex energies, and the strongest cases are similar between the two sets. They also correlate well with similar relative interaction energies derived directly from frequencies of bases in the bacteriophage lambda operator sequences. These results suggest that such potentials are general and that extensive combinatorial binding studies can be used to derive potential energies for DNA-protein interactions.     

Alterations of G-protein coupling function in phosphoinositide signaling pathways of cells transformed by ras and other membrane-associated and cytoplasmic oncogenes.

We showed previously that transformation by cytoplasmic and membrane-associated oncogenes including ras results in uncoupling between surface stimulation by platelet-derived growth factor, bombesin, and serum and activation of intracellular phospholipase C (PLC); this uncoupling does not involve alterations at the receptor or effector enzyme levels (T. Alonso, R. O. Morgan, J. C. Marvizon, H. Zarbl, and E. Santos, Proc. Natl. Acad. Sci. USA 85:4271-4275, 1988). In this study, we stimulated normal and oncogene-transformed NIH 3T3 cells with fluoroaluminate (AIF4-), thus directly activating PLC-associated G protein(s) and bypassing the receptor step. A1F4(-)-elicited PLC responses were significantly impaired in transformed cells when compared with those in their normal counterparts, suggesting that the uncoupling of PLC is the result, at least in part, of functional impairment at the G-protein level. Transformation by ras oncogenes has also been reported to result in enhanced PLC response to bradykinin resulting from increased receptor numbers (G. Parries, R. Hoebel, and E. Racker, Proc. Natl. Acad. Sci. USA 84:2648-2652, 1987; J. Downward, J. de Gunzburg, R. Riehl, and R. Weinberg, Proc. Natl. Acad. Sci. USA 85:5774-5778, 1988). We demonstrate here that transformation by other membrane-associated and cytoplasmic oncogenes also results in increased responsiveness to bradykinin ("supercoupling") and enhanced receptor numbers. However, there is no direct correlation between the number of receptors and the enhancement in responsiveness, suggesting that other factors besides receptor number are also involved in the enhanced responses. We propose that a common effect of transformation by cytoplasmic and membrane-associated oncogenes is functional alteration of coupling G proteins and that a similar modification of different kinds of G proteins may account for the pleiotropic alterations of signal transduction (uncoupling and supercoupling) observed.     

Cytosolic chloride ions stimulate Ca(2+)-induced exocytosis in melanotrophs.

We used the whole-cell patch-clamp technique to study the secretory activity of single cells by monitoring changes in membrane capacitance [Neher, E. and Marty, A. (1982) Proc. Natl. Acad. Sci. USA 79, 523-535] in anterior pituitary cells. Unexpectedly we have observed that increasing intracellular chloride ions stimulate Ca(2+)-induced exocytosis in a dose-dependent fashion (Kd = 12 mM). These results demonstrate a role of cytosolic chloride ions in the regulation of exocytotic secretion in anterior pituitary cells. It is suggest that chloride channels, in addition to playing a part in regulating membrane electrical activity [Korn, S.J., Bolden, A. and Horn, A. (1991) J. Physiol. 439, 423-437; Penner, R., Matthews, G. and Horn, A. (1988) Nature 334, 499-504] and cytosolic pH [Kaila, K. and Voipio, J. (1987) Nature 330, 163-165], are also involved in the modulation of cytosolic chloride concentration and thus in the control of exocytosis.     

Chromosomal locations of members of a family of novel endogenous human retroviral genomes.

Human cellular DNA contains two distinguishable families of retroviral related sequences. One family shares extensive nucleotide sequence homology with infectious mammalian type C retroviral genomes (T. I. Bonner, C. O'Connell, and M. Cohen, Proc. Natl. Acad. Sci. USA 79:4709-4713, 1982; M. A. Martin, T. Bryan, S. Rasheed, and A. S. Khan, Proc. Natl. Acad. Sci. USA 78:4892-4896, 1981). The other family contains major regions of homology with the pol genes of infectious type A and B and avian type C and D retroviral genomes (R. Callahan, W. Drohan, S. Tronick, and J. Schlom, Proc. Natl. Acad. Sci. USA 79:5503-5507, 1982; I. M. Chiu, R. Callahan, S. R. Tronick, J. Schlom, and S. A. Aaronson, Science 223:364-370, 1984). Analysis of the human recombinant clone HLM-2 has shown that the pol gene in the latter family is located within an endogenous proviral genome (R. Callahan, I. M. Chiu, J. F. H. Wong, S. R. Tronick, B. A. Roe, S. A. Aaronson, and J. Schlom, Science 228:1208-1211, 1985). We show that the proviral genome in HLM-2 and the related recombinant clone HLM-25 are located, respectively, on human chromosomes 1 and 5. Other related proviral genomes are located on chromosomes 7, 8, 11, 14, and 17.     

The actin-based nanomachine at the leading edge of migrating cells.

Two fundamental parameters of the highly dynamic, ultrathin lamellipodia of migrating fibroblasts have been determined-its thickness in living cells (176 +/- 14 nm), by standing-wave fluorescence microscopy, and its F-actin density (1580 +/- 613 microm of F-actin/microm(3)), via image-based photometry. In combination with data from previous studies, we have computed the density of growing actin filament ends at the lamellipodium margin (241 +/- 100/microm) and the maximum force (1.86 +/- 0.83 nN/microm) and pressure (10.5 +/- 4.8 kPa) obtainable via actin assembly. We have used cell deformability measurements (. J. Cell Sci. 44:187-200;. Proc. Natl. Acad. Sci. USA. 79:5327-5331) and an estimate of the force required to stall the polymerization of a single filament (. Proc. Natl. Acad. Sci. USA. 78:5613-5617;. Biophys. J. 65:316-324) to argue that actin assembly alone could drive lamellipodial extension directly.     

Immunological and structural homology between human T-cell leukemia virus type I envelope glycoprotein and a region of human interleukin-2 implicated in binding the beta receptor.

The N-terminal segment of human interleukin-2 (hIL-2) appears to mediate binding of the beta hIL-2 receptor (R. Robb, C. Rusk, J. Yodoi, and W. Greene, Proc. Natl. Acad. Sci. USA 84:2002-2006, 1987). An affinity-purified antibody prepared against this peptide segment (p81) is shown here to cross-react with a homologous region of the human T-cell leukemia virus type I (HTLV-I) envelope glycoprotein, raising the interesting possibility that the envelope glycoprotein of HTLV-I can interact with the beta hIL-2 receptor.     

Failure to transmit disease from gray tremor mutant mice.

Mice homozygous for mutant alleles at the gray tremor (gt) locus develop a marked non-intention tremor beginning at 8 days of age. Most homozygous mice die by 3 months. Homozygotes exhibit intense vacuolation of the central nervous system gray matter and vacuolation and hypomyelination of some white matter tracts. Based on neuropathological similarities with scrapie, other investigators inoculated wild-type mice with gray tremor brain homogenates to test the hypothesis of transmissibility. Published reports indicated that spongiform encephalopathy (R. L. Sidman, H. C. Kinney, and H. O. Sweet, Proc. Natl. Acad. Sci. USA 82:253-257, 1985) and disease, including hind limb paralysis in NFS mice (P. M. Hoffman, R. G. Rohwer, C. MacAuley, J. A. Bilello, J. W. Hartley, and H. C. Morse III, Proc. Natl. Acad. Sci. USA 84:3866-3870, 1987), were transmitted by inoculation of gt/gt brain homogenates. In our hands, however, no NFS/NCr animals inoculated intracerebrally with gt/gt or +/+ brain preparations showed any signs of disease or pathological changes in the brain. Positive transmission by other investigators may reflect the microbiological status of their donor or recipient mice.     

The yeast DNA repair proteins RAD1 and RAD7 share similar putative functional domains

Sequence information on eukaryotic DNA repair proteins provided so far only few clues concerning possible functional domains. Since the DNA repair process involves a strict sequential complex formation of several proteins [1988) FASEB J. 2, 2696-2701], we searched for special protein-protein interacting domains, which consist of tandemly repeated leucine rich motifs (LRM). Search algorithms, capable of detecting even largely divergent repeats by assessing their significance due to the tandem repetitivity, revealed that the yeast DNA repair proteins RAD1 and RAD7 contain 9 and 12 tandem LRM repeats, respectively. These results represent the first clues concerning specific domains in these proteins and assign them to the LRM superfamily, which includes such members as yeast adenylate cyclase, cell surface protein receptors and ribonuclease/angiogenin inhibitor, all exerting their function by specific protein-protein interactions involving LRM domains [( 1988) EMBO J. 7, 4151-4156; (1990) Proc. Natl. Acad. Sci. USA 87, 8711-8715; (1989) Science 245, 494-499; (1990) Mol. Cell. Biol. 10, 6436-6444; (1989) Proc. Natl. Acad. Sci. USA 86, 6773-6777].     

AGTR1 as a therapeutic target in ER-positive and ERBB-negative breast cancer cases

Comment on: Rhodes DR, et al. Proc Natl Acad Sci USA 2009; 106:10284-9.     

The dnaC protein of Escherichia coli. Purification, physical properties and interaction with dnaB protein.

E.coli dnaC protein was purified to near-homogeneity in using a dnaC complementation assay [S.Wickner, I.Berkower, M.Wright, and J.Hurwitz (1973) Proc. Natl. Acad. Sci. USA 70, 2369-2373]. Purification was achieved by taking advantage of the hydrophobic interaction of dnaC protein with aliphatic and aromatic matrixes and with Brij58 as stabilizing agent. A sedimentation coefficient for the dnaC protein of 2.6 S corresponding to a molecular weight of approximately 26,000 was estimated from glycerol gradient centrifugation. A polypeptide molecular weight of 28,000 was determined by densitometry on a denaturing gel. In the presence of ATP the dnaC protein forms a complex with dnaB protein [S.Wickner and J.Hurwitz (1975) Proc.Natl.Acad.Sci. USA 72, 921-925]. For the dnaB . dnaC complex a sedimentation coefficient of 14.5 S was measured by glycerol gradient centrifugation, indicating a molecular weight of about 400,000. The ratio of the dnaC and dnaB polypeptides in the complex is approximately 1, as determined on a denaturing gel. It is suggested that the complex consists of the dnaB protein hexamer and six dnaC polypeptides amounting to a calculated molecular weight of about 450,000.     

PARP-3, a DNA-dependent PARP with emerging roles in double-strand break repair and mitotic progression

Comment on: Boehler C, et al. Proc Natl Acad Sci USA 2011; 108:2783-8.     

MiRNA activity directs stem cell commitment to a particular fate

Comment on: Mann M, et al. Proc Natl Acad Sci USA. 2010; 107:15804-9.     

Do transcription factories and TOP2B provide a recipe for chromosome translocations in therapy-related leukemia?

Comment on: Cowell IG, et al. Proc Natl Acad Sci USA 2012; 109:8989-94.     

How a mismatch repair enzyme balances the needs for efficient lesion processing and minimal action on undamaged DNA

Comment on: Maiti A, et al. Proc Natl Acad Sci USA 2012; 109:8091-6.     

A small plasmid for recombination-based screening.

We reported recently the construction of the 4.4-kb R6K-derived pMAD1 plasmid carrying supF [Stewart et al., Gene 106 (1991) 97-101] that does not share nt sequences with ColE1 and therefore permits recombination-based screening of lambda libraries that contain ColE1 sequences. Here we describe the construction of the 2.5-kb R6K-derived plasmid, pMAD3, that lacks the pi-encoding pir gene required for R6K replication. To supply pi [Inuzuka and Helinski, Proc. Natl. Acad. Sci. USA 75 (1978) 5381-5385] in trans, we employed pPR1 delta 22pir116, referred to henceforth as pPR1 [McEachern et al., Proc. Natl. Acad. Sci. USA 86 (1989) 7942-7946; Dellis and Filutowicz, J. Bacteriol. 173 (1991) 1279-1286]. Plasmid pMAD3 is small enough to be amplified readily by PCR [Saiki et al., Science 230 (1985) 1350-1354]. This permits the insertion of larger fragments and the retrieval of larger lambda inserts, as well as the use of a simplified PCR-based cloning protocol which utilizes annealing rather than ligation to create recombinants in pMAD3 [Nisson et al., PCR Methods and Applications 1 (1991) 120-123].     

A simple and efficient method for the separation and detection of small DNA fragments by electrophoresis in formamide containing agarose gels and Southern blotting to DBM-paper.

We show in this report that DNA fragments smaller than 300 bp are separated with high resolution by electrophoresis in concentrated (up to 7%) agarose gels containing 50% formamide. The separated DNA fragments can subsequently be quantitatively transferred to DBM-paper [Alwine, J.C. et al., Proc. Natl. Acad. Sci. USA (1977) 74, 5350-54] using the Southern technique [Southern, E.M., J. Mol. Biol. (1975) 98, 503-517], while preserving the sharpness of the original gel pattern. Since thin (0.2-0.4 mm) and thick (up to 5 mm) agarose slab gels can be easily handled in vertical or horizontal apparatus, this method should prove to be a very useful extention of the Southern technique, applicable to a variety of analytical and preparative purposes.